Method and substances for isolation and detection of small polynucleotides

ABSTRACT

A capture probe suitable for use with methods for isolating, labeling or detecting small polynucleotides. A method for isolating a small polynucleotide of interest from a sample comprising hybridizing the small polynucleotide to the capture probe and lengthening the small polynucleotide by primer extension or ligation. A method for detecting a small polynucleotide of interest following isolation by amplification of the primer extension products and/or hybridization and subsequent cleavage of dual labeled detector probes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 11/958,180, titled “Methods and Substances for Isolation andDetection of Small Polynucleotides” filed Dec. 17, 2007, which is acontinuation-in-part of International Patent Application No.PCT/US07/77311, titled “Method and Substances for Isolating RNAs,” filedAug. 30, 2007, which claims the benefit of U.S. Provisional PatentApplication No. 60/824,068, titled “Method and Substances for IsolatingRNAs,” filed Aug. 30, 2006; and claims the benefit of U.S. ProvisionalPatent Application No. 60/825,888, titled “Method and Substances forIsolating RNAs,” filed Sep. 15, 2006; and claims the benefit of U.S.Provisional Patent Application No. 60/863,886, titled “Method andSubstances for Isolating RNAs,” filed Nov. 1, 2006; and claims thebenefit of U.S. Provisional Patent Application No. 60/866,210, titled“Method and Substances for Isolating RNAs,” filed Nov. 16, 2006; andclaims the benefit of U.S. Provisional Patent Application No.60/871,094, titled “Method and Substances for Isolating RNAs,” filedDec. 20, 2006; the contents of which are incorporated in this disclosureby reference in their entirety.

BACKGROUND

There are a large variety of small polynucleotides, both naturallyoccurring and synthetic, which are of scientific or commercial interest.Exemplary small polynucleotides include microRNAs, small nucleolar RNAs,short interfering RNAs (natural or synthetic), guide RNAs, nuclear RNAs,ribosomal RNAs, transfer RNAs as well as small antisense DNAs or smallpolynucleotide degradation products. Of particular interest aremicroRNAs (miRNAs), naturally occurring, single strandedpolyribonucleotides (polyRNAs) of between 18 and 24 RNA residues, whichare derived from a longer, naturally occurring noncoding eukaryoticprecursor RNA transcript (usually having a ‘hairpin’ configuration), andmiRNAs play a significant role in cellular developmental anddifferentiation pathways. Consequently, there have been considerableefforts made to understand and characterize the temporal, spatial andcellular expression levels and patterns of expression of miRNAs toascertain their precise role in cellular development and differentiationin both normal and disease states.

miRNAs are currently studied by, first, obtaining the total RNA from asample. Next, the total RNA is fractionated into subpopulations by gelelectrophoresis or by chromatographic fractionation and size selectiveelution. Then, the appropriate section of the gel is cut, and the 18-24RNAs are eluted from the gel, or the eluted fraction containing singlestranded RNA's in the size range of 18-24 ribonucleotides is collected,usually the RNA fraction of less than 500-200 nucleotides in length.Next, the RNAs are isolated by precipitation and the miRNAs arecharacterized.

However, these methods are disadvantageous because they do not work wellwhen the amount of sample is small, such as samples from tumor tissue orbiopsy material. Further, characterization of the miRNAs isolated bypresent methods usually comprises a several step amplification procedurefollowed by detection, quantitation, cloning and sequencing. Because ofthe large number of steps in these processes, and the notoriousinefficiencies associated with the repeated purification, isolation andidentification of miRNAs, it is time consuming, relatively expensive,requires relatively large amounts of material and is not fullyrepresentative of the population of miRNAs expressed within a sample,such as within a tumor, or those miRNAs expressed in low abundance.Additionally, the methods are not specific to isolating and identifyingmiRNAs, and often isolate and identify siRNA, tRNA, 5S/5.8S rRNA anddegraded RNA from additional cellular RNAs.

Therefore, there is the need for an improved method for isolation andidentification of miRNAs, other small regulatory RNAs and shortinterfering RNAs (siRNAs) that is not associated with thesedisadvantages.

SUMMARY

According to one embodiment of the invention, there is provided acapture probe for use in isolating and detecting small polynucleotides.The capture probe is a polynucleotide that includes a spacer segmenthaving a spacer segment sequence, the spacer segment having a 3′ end anda 5′ end; a template segment having a template segment sequence, thetemplate segment having a 3′ end and a 5′ end; and a smallpolynucleotide binding segment having a small polynucleotide bindingsegment sequence. The 5′ end of the spacer segment is connected to the3′ end of the small RNA binding segment; and the 3′ end of templatesegment is connected to the 5′ end of the small RNA binding segment

The small polynucleotide binding segment is substantially complementaryto, and capable of hybridizing to, one or more than one smallpolynucleotides of interest by Watson-Crick base pairing. In preferredversions of the capture probe the small polynucleotide of interest isselected from the group consisting of miRNA (microRNA), snoRNA (smallnucleolar RNA), and siRNA (short interfering RNA; small interfering RNA;silencing RNA).

In one embodiment the capture probe further comprises a solid phasebinding segment of a molecular composition capable of binding to a solidphase.

In one embodiment of the capture probe the spacer segment includes a RNApolymerase termination site.

In a preferred embodiment of the capture probe the small polynucleotidebinding segment is substantially complementary to, and capable ofhybridizing to a miRNA of interest.

In one embodiment of the capture probe the template segment includes oneor more than one sequence comprising a polynucleotide polymeraserecognition site or a sequence that is complementary to a polynucleotidepolymerase recognition site.

In one embodiment of the capture probe the template segment includes oneor more than one sequence that is a restriction enzyme recognitionmotif.

In one embodiment of the capture probe the template segment includes oneor more than one sequence that is complementary to a RNA-cleavingcatalytic nucleic acid or DNAzyme.

One embodiment of the present invention provides a composition comprisedof two or more capture probes. The composition includes (a) a firstcapture probe having a first spacer segment, a first smallpolynucleotide binding segment and a first template segment; and (b) asecond capture probe having a second spacer segment, a second smallpolynucleotide binding segment and a second template segment, where thesecond small polynucleotide binding segment has a different smallpolynucleotide binding segment sequence than the first smallpolynucleotide binding segment and the second template segment has adifferent template segment sequence than the first template segment.

Another embodiment of the present invention provides a method ofisolating a small polynucleotide of interest. The method includes thesteps of (a) providing one or more than one capture probe as set forthabove; (b) providing a sample comprising a small polynucleotide ofinterest (c) combining the capture probe and the sample; (d) allowingthe small polynucleotide of interest to hybridize with the smallpolynucleotide binding segment of the capture probe to form a smallpolynucleotide/capture probe complex; (e) combining the smallpolynucleotide/capture probe complex with a polynucleotide polymerase,preferably a polymerase capable of using RNA as a primer, and a set ofnucleotide triphosphates; and (f) extending the hybridized smallpolynucleotide of interest to form an extension product, where theextension product comprises the small polynucleotide of interestconnected at the 3′ end to an extended segment, the extended sequencecomprising a sequence complementary to the template segment of thecapture probe, and where the extension product is hybridized to thecapture probe to form an extension product/capture probe complex.

In preferred versions of the method the small polynucleotide of interestis selected from the group consisting of miRNAs, snoRNAs, or siRNAs. Ina particularly preferred version, the small polynucleotide of interestis a miRNA.

In one embodiment of the method, the capture probe also contains a solidphase binding segment and the small polynucleotide/capture probe complexor the extension product/capture probe complex is captured to a solidphase by binding of capture probe to a solid support via the solid phasebinding segment.

Another embodiment provides a method for detecting a smallpolynucleotide of interest from a sample, which includes the steps of:(a) isolating a small polynucleotide of interest as set forth above,where the capture extension probe is attached to a fluorescent bead andthe extended segment contains one or more labeled nucleotide residues;and (b) detecting the fluorescent bead and the labeled extension producthybridized to the capture extension probe.

Another embodiment provides a method of detecting a small polynucleotideof interest, which includes the steps of: (a) isolating a smallpolynucleotide of interest as set forth above, wherein the templatesegment of the capture probe contains one strand of an RNA polymeraserecognition sequence and the extension step forms a double stranded RNApolymerase promoter; (b) combining the extension product/capture probecomplex with a RNA polymerase that recognizes the double stranded RNApolymerase promoter; and (c) transcribing the sequences downstream fromthe promoter to synthesize a single stranded RNA product containing asmall RNA binding sequence. In a preferred embodiment of the detectionmethod, the spacer segment of the capture probe contains an RNApolymerase stop site. In another preferred embodiment, the methodfurther comprises repeating the transcription step one or more times.

One embodiment provides another method for detecting a small RNA ofinterest in a sample, which includes the steps of: (a) isolating thesmall polynucleotide of interest as set forth above; (b) providing aligase and a linker segment, the linker segment comprising apolynucleotide having 3′ end and a 5′ end, the linker segment having alinker segment sequence, wherein the linker segment sequence issubstantially complementary to, and capable of hybridizing to, thespacer segment sequence by Watson-Crick base pairing; (c) allowing thelinker segment to hybridize to the spacer segment; and (d) ligating the3′ end of the linker segment to the 5′ end of the small RNA of interestto form a ligated extension product substantially complementary to, andcapable of hybridizing to, the capture probe sequence. A preferredversion of the method further comprises amplifying the ligated extensionproduct and the capture probe by a polymerase chain reaction.

Another embodiment provides a method of detecting a small polynucleotideof interest, which includes the steps of (a) providing a dual-labeleddetector probe, having one label attached to the 5′ end of detectorprobe molecule, another label attached to the 3′ end of the detectorprobe, and a detector probe sequence that is substantially complementaryto, and capable of hybridizing to a detector probe binding sequencewithin the template segment of the capture probe; (b) isolating a smallpolynucleotide of interest as set forth above, where (1) combining thecapture probe and sample further comprises adding the dual-labeleddetector probe to the combination; (2) allowing the detector probe tohybridize with the detector probe binding sequence of the capture probeor small polynucleotide/capture probe complex; (3) adding a polymerasehaving 5′ to 3′ exonuclease activity and nucleotide mix to thehybridized detector probe and small polynucleotide/capture probe complexso that the detector probe is hydrolyzed by the 5′ to 3′ exonuclease ofthe polynucleotide polymerase; and (4) detecting the change influorescence properties of one or more of the labels followinghydrolysis of the detector probe.

One embodiment provides another method of detecting a smallpolynucleotide of interest, including the steps of (a) isolating a smallpolynucleotide of interest as set forth above, where (1) the templatesegment comprises one or more than one sequence that is one strand of adouble stranded restriction enzyme recognition motif; and (2) theextension step converts the single stranded restriction enzymerecognition sequence contained within the template segment of thecapture probe into a double stranded restriction enzyme recognitionsequence. The method further comprises (b) providing a restrictionenzyme that recognizes and acts upon the restriction enzyme recognitionsequence of the extended segment; (c) contacting the restriction enzymewith the restriction enzyme recognition sequence; (d) nicking theextension product at or near the restriction enzyme recognition sequenceof the extended segment to produce a 3′ ended fragment containing thesmall polynucleotide of interest and a 5′ ended nicked extensionfragment; and (e) displacing and detecting the nicked extensionfragment. In one version of the method the restriction enzymerecognition motif is recognized by a nicking endonuclease. In anotherversion the restriction enzyme recognition motif of the template segmentcontains one or more than one nucleotide analogue, which rendersrestriction enzyme recognition motif of the template segment resistantto the endonuclease activity of the restriction enzyme. In a preferredversion, the method further comprises cycles of extending the 3′-endedfragment containing the hybridized small polynucleotide of interest witha polymerase such that the restriction enzyme recognition motif isrejuvenated and the 5′-ended nicked extension fragment is displaced.

In another embodiment of the detection method (a) the template segmentof the capture probe comprises a first restriction site and a secondrestriction site, where the first restriction site differs from thesecond restriction site, (b) the extension step converts the firstrestriction site into a double stranded restriction enzyme recognitionsequence capable of being nicked on the extended segment, but not thetemplate segment, and the second restriction site is converted into asecond double stranded restriction recognition sequence; (c) the nickingstep comprises contacting the extension product/capture probe complexwith a nicking agent which recognizes and acts on the first restrictionsite, but not the second restriction site, such that the extensionproduct is selectively nicked at or near the first restriction site ofthe extended segment to produce a nicked extension fragment. Thedetecting step then comprises: (1) providing an dual labeled detectorprobe, which is complementary to and capable of hybridizing to thenicked extension fragment; (2) hybridizing the probe to the nickedextension fragment to form a double stranded probe/nicked extensionfragment complex; (3) contacting the double stranded probe/nickedextension fragment complex with a nicking agent capable of recognizingand nicking the detector probe sequence; and (4) detecting a change influorescence associated with nicking the dual labeled detector probe. Ina preferred version of this method, the first restriction enzymerecognition motif is recognized by a nicking endonuclease. In anotherversion of this method, the second restriction enzyme recognition motifof the template segment contains one or more than one nucleotideanalogue, which renders the restriction enzyme recognition motif of thetemplate segment resistant to the endonuclease activity of therestriction enzyme.

In another embodiment of the detection method (a) the template segmentfurther comprises one or more than one DNAzyme complementary sequencethat is complementary to a DNAzyme motif, a first flanking segment and asecond flanking segment, the first flanking segment flanking the 5′ endof the DNAzyme complementary sequence and the second flanking segmentflanking the 3′ end of the DNAzyme complementary sequence; and (b) thedisplacement of the nicked extension fragment provides a functionalDNAzyme capable of hybridizing to and cleaving a suitable substrateprobe at a DNAzyme cleavage site. The detecting step further comprises(1) providing suitable substrate probe comprising an RNA polynucleotideor a chimeric RNA/DNA polynucleotide, the substrate probe having onelabel attached to the 5′ end of the substrate probe molecule and anotherlabel attached to the 3′ end of the substrate probe, the substrate probecomprising a first substrate probe segment having a first substrateprobe sequence, a DNAzyme cleavage site and a second substrate probesegment having a second substrate probe sequence, where first substrateprobe sequence of the substrate probe is substantially identical to thefirst flanking segment of the template segment and the second substrateprobe sequence is substantially identical to the second flankingsequence of the template segment; (2) contacting the substrate probe andthe nicked extension fragment such that a loop structure containing theDNAzyme motif is formed in the nicked extension fragment by Watson-Crickbase pairing between the first substrate probe sequence andcomplementary sequences contained within the nicked extension fragmentand between the second substrate probe and complementary sequencescontained within the nicked extension fragment; (3) cleaving thesubstrate probe at the DNAzyme cleavage site; and (4) detecting a changein fluorescence associated with cleaving the substrate probe.

One embodiment provides a kit for the isolation and detection of smallRNAs, which can include (1) an equimolar mix of capture probes; (2) anucleotide mix containing deoxyribonucleotide triphosphates orribonucleotide triphosphates; (3) a polymerase; (4) streptavidin coatedparamagnetic beads; (5) one or more than one dual labeled detectorprobe, the detector probe having a detector probe sequence that issubstantially complementary to, and capable of hybridizing to a detectorprobe binding sequence within the template segment of the captureprobes; (6) a ligase enzyme; (7) an oligonucleotide linker that issubstantially complementary to and capable of hybridizing to the spacersegment of the capture probes; and/or (8) one or more than restrictionenzyme specific for a restriction enzyme recognition sequence containedin the capture probes The invention is described in more detail by thefollowing description.

DRAWINGS

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying figures where:

FIGS. 1A-1D are schematic diagrams of some of the steps in certainembodiments of a method of isolating and detecting miRNAs and othersmall polynucleotides using a capture probe according to the presentinvention;

FIGS. 2A-2D are other schematics of some of the steps in certainembodiments of a method of isolating and detecting miRNAs and othersmall polynucleotides using one version of the capture probe having anRNA polymerase recognition site according to the present invention;

FIGS. 3A-3B show diagrams of some of the steps in certain otherembodiments of a method for isolating and detecting miRNAs or othersmall polynucleotides using a capture probe to guide ligation of alinker according to the present invention;

FIGS. 4A-4B show diagrams of some of the steps in other embodiments of amethod for detecting miRNAs or other small polynucleotides using acapture probe and a detector probe according to the present invention;

FIGS. 5A-5D show diagrams of some of the steps in other embodiments of amethod for isolating and detecting miRNAs or other small polynucleotidesusing another version of the capture probe that generates a nick siteaccording to the present invention.

FIGS. 6A-6C show diagrams of some of the steps in other embodiments of amethod for detecting miRNAs or other small polynucleotides using anotherversion of the capture probe and a detector probe according to thepresent invention.

FIGS. 7A-7C show diagrams of some of the steps in other embodiments of amethod for isolating and detecting miRNAs or other small polynucleotidesusing another version of a capture probe capable of generating a DNAzymeaccording to the present invention.

FIG. 8 is a scanned image of the hybridized GenoExplorer miRNA chipusing 10 μm scan resolution and a 70% laser gain; and

FIG. 9 is a graph of fluorescent intensity for the sample of 12different miRNAs detected using a method according to the presentinvention.

DESCRIPTION

According to one embodiment of the present invention, there is provideda method for isolating small polynucleotides, such as for example miRNAs(small RNAs), short interfering RNAs and other small regulatory RNAs andDNAs. According to another embodiment of the present invention, there isprovided a method for identifying small polynucleotides of interest. Inone embodiment, the method for identifying small polynucleotides ofinterest comprises, first, isolating the small polynucleotides ofinterest according to the present invention. According to anotherembodiment of the present invention, there is provided one or more thanone capture probe and one or more than one set of capture probessuitable for use with a method for isolating small polynucleotides. Inone embodiment, the method for isolating small polynucleotides is amethod according to the present invention. The method and capture probeswill now be disclosed in detail.

As used in this disclosure, except where the context requires otherwise,the term “comprise” and variations of the term, such as “comprising,”“comprises” and “comprised” are not intended to exclude other additives,components, integers or steps.

As used in this disclosure, the term “small RNAs” means a naturallyoccurring, single stranded RNA of between 18 and 24 RNA residues,usually with a 5′ terminal phosphate group, usually referred to as“mature micro RNAs,” which is derived from a larger naturally occurringprecursor RNA, usually having a “hairpin” configuration.

As used in this disclosure the terms “small polynucleotide” and “smallpolynucleotides” refer to polynucleotides which are between 17 and 200residues in length, usually single stranded RNA or DNA, whichencompasses the group of noncoding regulatory RNAs including for examplemiRNAs, snoRNAs, snRNAs, siRNAs, antisense DNAs and Okazaki fragments.

As used in this disclosure, the terms “one or more than one smallpolynucleotides,” “a small polynucleotide” and “the smallpolynucleotide” are intended to be synonymous, that is are intended toindicate either one small polynucleotide of interest or a plurality ofsmall polynucleotides of interest, except where the context requiresotherwise.

As used in this disclosure, the terms “one or more than one captureprobe,” “a capture probe,” “the capture probe,” “the capture probes,”“capture-extension probe,” “capture-extension probes,” “capture andextension template probe” and “capture and extension template probes”are intended to be synonymous, and are intended to indicate either thesingular or plural, except where the context requires otherwise.

As used in this disclosure, the term “substantially complementary” andvariations of the term, such as “substantial complement,” means that atleast 90% of all of the consecutive residues in a first strand arecomplementary to a series of consecutive residues of the same length ofa second strand. As will be understood by those with skill in the artwith reference to this disclosure, one strand can be shorter than theother strand and still be substantially complementary. With respect tothe invention disclosed in this disclosure, for example, the smallpolynucleotide or small polynucleotide binding segment can be shorter orlonger than the complementary small polynucleotide of interest; however,it is preferable that the small polynucleotide binding segment is of thesame length and is substantially complementary to its correspondingsmall polynucleotide.

As used in this disclosure, the term “hybridize” and variations of theterm, such as “hybridizes” and “hybridized,” means a Watson-Crick basepairing of complementary nucleic acid single strands or segments ofstrands to produce an anti-parallel, double-stranded nucleic acid, andas used in this disclosure, hybridization should be understood to bebetween substantially complementary strands unless specified otherwise,or where the context requires otherwise. As an example, hybridizationcan be accomplished by combining equal molar concentrations of each ofthe pairs of single strands, such as 100 pmoles, in the presence of 5 μgyeast tRNA (transfer RNA) in a total volume of 50 μl of aqueous buffercontaining 400 mM MOPS, 80 mM DTT, and 40 mM MgCl2 at a pH of 7.3, andthen incubating the mixture at 25° C. for one hour while shaking gently.

As used in this disclosure, the term “near the end” and variations ofthe term, means within 20% of the residues of the identified endresidue. For example, near the end of a 20 residue strand, means thefirst four residues of the identified 5′ or 3′ end or terminus end ofthe strand.

As used in this disclosure, the terms “extension” or “extensionreaction” indicates the extension of the 3′ end of a polynucleotide bythe action of a polymerase in conjunction with all the accessoryreagents and conditions for this reaction to occur.

Capture Probes

According to one embodiment of the present invention, there is provideda capture probe 10 suitable for use with a method for isolating smallRNAs or DNAs. Referring to FIG. 1A, the capture probe comprises from its3′ end to its 5′ end covalently joined or connected segments: a) a solidphase binding segment 20, b) a spacer segment 30, and c) a smallpolynucleotide binding segment 40 having a small polynucleotide bindingsegment sequence, where the small polynucleotide binding segment issubstantially complementary to and capable of hybridizing to one or morethan one small polynucleotide of interest by Watson-Crick base pairing,and d) a template segment 50.

In one embodiment, the capture probe 10 comprises a substance selectedfrom the group consisting of one or more than one type ofpolynucleotide, one or more than one of polynucleotide analog, and acombination of one or more than one type of polynucleotide andpolynucleotide analog.

In one embodiment the capture probe comprises a solid phase bindingsegment 20 of a molecular composition capable of binding to a solidphase, such as for example biotin coupled to the 3′ end of the captureprobe and its ability for binding to avidin or streptavidin immobilizedto a solid phase, such as for example streptavidin coated paramagneticparticles or streptavidin coated wells of a microtiter plate. In anotherembodiment, the solid phase binding segment 20 is a substance capable ofcovalent binding to a solid phase, such as for example a primary aminecoupled to carboxylic acid groups on a solid phase using carbodiimideactivation and amide bond formation in between the primary amine of thesolid phase binding segment and the carboxylic acid groups on the solidphase. Other suitable methods of covalent coupling of polynucleotides tosolid phases are well known in the art. In one embodiment, the solidphase binding segment 20 is either the 3′, 5′ or both ends of thecapture probes 10, they may also be interior to either the spacersegment 30 or the template segment 50 or both segments of the captureprobes 10. Further the solid phase binding segment 20 can be addedduring the synthesis of the capture-extension probes 10, for example asa biotin phosphoramidite during polynucleotide synthesis as will beunderstood by those skilled in the art. In addition the solid phasebinding segment 20 can be introduced after the synthesis of the acontiguous capture probe containing the spacer segment 30, the smallpolynucleotide binding segment 40 and the template segment 50, forexample by the incorporation of a biotin labeled dUTP to the 3′ terminusof the capture probe by the action of terminal transferase usingbiotinylated dUTP as the source for biotin.

The spacer segment 30 of the capture probe comprises a polynucleotidesequence, having a predetermined sequence or predetermined size,designed to provide one or more functional features. In one embodiment,the spacer segment is of sufficient length to minimize steric hindranceof hybridization complexes forming with the polynucleotide bindingsegment. In one embodiment the spacer segment includes a primer bindingsite for amplification reactions. In another embodiment, shown in FIG.3A, the spacer segment includes a docking site for a linker in ligationreactions. In yet another embodiment, the spacer includes one or morethan one desired restriction enzyme recognition site. In anotherembodiment, the spacer includes a RNA polymerase recognition site. Inone embodiment, shown in FIG. 2A, the spacer includes a transcriptiontermination site 36.

The polynucleotides of the spacer segment 30 may be naturally occurring,synthetic or nucleotide analogs comprising 5-50 nucleotides, or 5-40nucleotides, preferably 5-30 nucleotides. In one embodiment, the spacersegment 30 consists of RNA. In one embodiment, the spacer segment 30consists of DNA. In one embodiment, the spacer segment 30 consists ofpolynucleotide analogs. In one embodiment, the spacer segment 30consists of a chimera of more than one polynucleotide or polynucleotideanalog selected from the group consisting of RNA, DNA, polynucleotideanalogs of RNA, and polynucleotide analogs of DNA. In anotherembodiment, the spacer segment 30 of the capture probe 10 comprises anorganic substance having a carbon backbone of 6-100 carbon atoms orother backbone configurations, for example polyethylene glycols with3-33 repeat units, or amides such as those comprised of amino caproicacid repeat units of 1-17 elements.

The small polynucleotide binding segment 40 is designed to form ahybridization complex with a polynucleotide of interest. In oneembodiment, the small polynucleotide of interest is a small RNAmolecule. In one embodiment, the small polynucleotide of interest is asmall DNA molecule. In one embodiment, the small polynucleotide bindingsegment 40 consists of between 18 and 24 DNA residues. In anotherembodiment, the small polynucleotide binding segment 40 consists of 18or 19 or 20 or 21 or 22 or 23 or 24 DNA residues. In another embodimentthe small polynucleotide binding segment 40 comprises a DNA of between17 and 100 polynucleotides. In another embodiment the smallpolynucleotide binding segment 40 comprises a DNA of between 17 and 60polynucleotides. In another embodiment the small polynucleotide bindingsegment 40 comprises between 17 and 40 polynucleotides.

The small polynucleotide binding segment 40 is substantiallycomplementary to, and capable of hybridizing to, one or more than onesmall polynucleotide of interest by Watson-Crick base pairing, includinga small polynucleotide of interest having a predetermined sequence orhaving a predetermined size, from a sample comprising substances thatare chemically related, such as for example, a mixture of messengermRNAs (messenger RNAs), tRNAs (transfer RNAs), rRNAs (ribosomal RNAs)and genomic DNA. A small polynucleotide of interest 60 can be selectedfrom any known small RNA from any suitable source, as will be understoodby those with skill in the art with reference to this disclosure. In oneembodiment, the small polynucleotide of interest 60 is selected from apublic database. In a preferred embodiment, the small polynucleotide ofinterest 60 is a miRNA and the public database is a central repositoryprovided by the Sanger Institute http://miRNA.sanger.ac.uk/sequences/ towhich newly discovered and previously known miRNA sequences can besubmitted for naming and nomenclature assignment, as well as placementof the sequences in a database for archiving and for online retrievalvia the world wide web. Generally, the data collected on the sequencesof miRNAs by the Sanger Institute include species, source, correspondinggenomic sequences and genomic location (usually chromosomalcoordinates), as well as full length transcription products andsequences for the mature fully processed miRNA.

To select the sequence or sequences of the small polynucleotide bindingsegment 40, when the target small RNAs comprise miRNA, a miRNA ofinterest or set of miRNAs of interest are selected from a suitablesource, such as for example, the Sanger Institute database or othersuitable database, as will be understood by those with skill in the artwith reference to this disclosure. If a set of miRNAs of interest isselected from a source that contains duplicate entries for one or morethan one miRNAs, in a preferred embodiment, the duplicated entries arefirst removed so that the set of sequences of miRNAs of interestcontains only one sequence for each miRNA of interest. In oneembodiment, the set of miRNAs of interest consists of one of each miRNAsfrom a single source or database, such as one of each miRNAs listed inthe central repository provided by the Sanger Institute.

In another embodiment the small polynucleotide of interest 60 is aeukaryotic small RNA. In another embodiment the small RNA of interest isa primate small RNA. In another embodiment the small RNA of interest isa virus small RNA. In a preferred embodiment, the small RNA of interestis a human small RNA. In another embodiment, the set of small RNAs ofinterest are all eucaryotic miRNAs. In another embodiment, the set ofsmall RNAs of interest are all primate miRNAs. In another embodiment,the set of small RNAs of interest are all human miRNAs.

In another embodiment the small polynucleotide of interest 60 is aeukaryotic small DNA. In another embodiment the small DNA of interest isa primate small DNA. In another embodiment the small DNA of interest isa virus small DNA. In a preferred embodiment, the small DNA of interestis a human small DNA. In another embodiment, the set of small DNAs ofinterest are all eucaryotic DNAs. In another embodiment, the set ofsmall DNAs of interest are all primate DNAs. In another embodiment, theset of small DNAs of interest are all human DNAs.

Next, the small polynucleotide binding segment 40 is selected to be thesubstantial complement of the small polynucleotide of interest sequence60. In a preferred embodiment, the small polynucleotide binding segment40 is exactly the complement to the small polynucleotide of interest 60in both length and sequence. In another embodiment, the smallpolynucleotide binding segment is a more than 90% complementary to asegment of the small polynucleotide of interest of the same length asthe small polynucleotide of interest sequence. In another embodiment,the small polynucleotide binding segment 40 is more than 80%complementary to a segment of the small polynucleotide of interest 60 ofthe same length as the small polynucleotide of interest sequence 60.

In one embodiment, the small polynucleotide binding segment 40 consistsof RNA. In one embodiment, the small polynucleotide binding segment 40consists of DNA. In one embodiment, the small polynucleotide bindingsegment 40 consists of polynucleotide analogs. In one embodiment, thesmall polynucleotide binding segment 40 consists of a chimera of morethan one polynucleotide or polynucleotide analog selected from the groupconsisting of RNA, DNA, polynucleotide analogs of RNA, andpolynucleotide analogs of DNA.

Additionally, the small polynucleotide binding segment 40 can becomplementary to miRNAs, snoRNAs, or siRNAs thereby facilitating theirassay.

Table I provides a list of sample small polynucleotide binding segments40 which consist of DNA along with the miRNAs that are the exactcomplement of the small RNA binding segments 40. As will be understoodby those with skill in the art with reference to this disclosure, othersmall RNA binding segments 40 will also be useful, including for examplesmall RNA binding segments 40 that are the cDNA of the small RNA bindingsegments 40 listed in Table I.

TABLE I EXAMPLES OF 8 SMALL RNA BINDING SEGMENTS FOR HUMAN MIRNASSmall RNA binding segment as DNA MICRO RNA polynucleotide SEQ ID NO:hsa-let-7a AACTATACAACCTACTACCTCA SEQ ID NO: 1 hsa-let-7eACTATACAACCTCCTACCTCA SEQ ID NO: 2 hsa-miR- GCTACCTGCACTGTAAGCACTTTSEQ ID NO: 3 106a hsa-miR- CGCGTACCAAAAGTAATAATG SEQ ID NO: 4 126*hsa-miR- TCACATAGGAATAAAAAGCCATA SEQ ID NO: 5 135a hsa-miR-GATTCACAACACCAGCT SEQ ID NO: 6 138 hsa-miR- CGAAGGCAACACGGATAACCTASEQ ID NO: 7 154 hsa-miR- AATAGGTCAACCGTGTATGATT SEQ ID NO: 8 154*

The template segment 50 of the capture probe comprises a polynucleotidesequence, having a predetermined sequence or predetermined size,designed to provide one or more functional features.

In a particularly preferred embodiment, the polynucleotide comprisingthe template segment 50 of the capture probe can serve as a template forthe synthesis of a complementary polynucleotide strand by the action ofa polynucleotide polymerase.

In one embodiment, shown in FIGS. 4A and 4B, the template segmentincludes a binding site for a detector probe 52.

In another embodiment, shown in FIG. 2A, the template segment includes apolynucleotide polymerase recognition site 54 or that is complementaryto a polynucleotide polymerase recognition site. In a preferredembodiment, the polynucleotide polymerase recognition site 54 is a motiffor a polynucleotide synthesis promoter selected from the groupconsisting of T7, SP6, a T3 DNA dependent RNA polymerase, a type 2 RNApolymerase of E. coli and single stranded DNA dependent N4 RNApolymerase. The polynucleotide synthesis promoter motif can be a motiffor any other suitable polynucleotide synthesis promoter, however, aswill be understood by those with skill in the art with reference to thisdisclosure.

In one embodiment the template segment includes a transcriptiontermination site.

In another embodiment, shown in FIG. 5A, the template segment comprisesone or more than one sequence that is a restriction enzyme recognitionmotif 56. In a particularly preferred embodiment, the specificrestriction enzyme recognition motif 56, when present, is not present inthe DNA analog of the miRNA or other small polynucleotide of interestthat is being isolated and identified by the present methods. In a oneembodiment, the restriction enzyme recognition motif 56, when present,is recognized by a nicking endonuclease. In a preferred embodiment therestriction site motif 56 of the template segment is not cut by thenicking endonuclease. In a particularly preferred embodiment, therestriction site 56 is recognized by a nicking endonuclease, such asN.BbvCI, N.AlwI, N.BstNBI, N.Bpu10I and the like available from NewEngland Biolabs (Ipswich, Mass., US). In one embodiment, the restrictionenzyme recognition motif 56 is recognized by a restriction enzymeselected from the group consisting of BamHI, Hind III and EcoR I. In apreferred embodiment, the restriction site motif 56 is recognized by arestriction enzyme selected from the group consisting of Not I, Xho I,Xma I and Nhe I, because BamH I, Hind III and EcoR I also act upon someDNA equivalents of sequences of miRNA. In a preferred embodiment, therestriction enzyme recognition motif 56 contains one or more than onemodified nucleotide or nucleotide analogue, which protects the templatesegment from the endonuclease activity of the restriction enzyme. Forexample, the restriction enzyme recognition motif 56 of the templatesegment 50 may contain one or more internucleoside bonds resistant tohydrolysis, such as phosphorothioate, boranophosphate,methylphosphonate, or peptide bonds. An alternative example of anucleotide analogue would be where a deoxyuridine is substituted for adeoxythymidine in a restriction enzyme recognition motif 56. As will beunderstood by those with skill in the art with reference to thisdisclosure, however, other suitable restriction site motifs can also beused.

In yet another embodiment, shown in FIG. 7A, the template segment 50contains one or more than one sequence 130 complementary to a DNAzyme.Examples of a RNA-cleaving DNA enzyme (DNAzyme) include the “10-23” andthe “8-17” general purpose RNA-cleaving DNA enzymes, which both containconserved catalytic sequences (GGCTAGCTACAACGA and TCCGAGCCGGACGA,respectively). The conserved catalytic domain is flanked by variablebinding domains capable of hybridizing to a target RNA by Watson-Crickbase pairing. Hybridization of the flanking binding domains to a targetRNA results in a loop structure containing the catalytic domain.Cleavage by an exemplary “10-23” DNAzyme occurs at a purine-pyrimidinedinucleotide of the target RNA, whereas cleavage by an exemplary “8-17”DNAzyme can occur at an AG dinucleotide of the target RNA. Accordingly,a sequence 130 complementary to a DNAzyme motif in accordance with thepresent embodiment will contain sequences complementary to a conservedcatalytic sequence, as will be understood by one of skill in the artwith reference to the present disclosure.

In one embodiment, the template segment 50 of the capture probecomprises a polynucleotide comprised of nucleotides which are naturallyoccurring, synthetic or nucleotide analogues.

In one embodiment, the template segment 50 comprises 1-50 nucleotides,in another embodiment the template segment comprises 1-40 nucleotides,and in yet another embodiment the template segment comprises 1-30nucleotides.

In one embodiment, the template segment 50 consists of RNA. In oneembodiment, the template segment 50 consists of DNA. In one embodiment,the template segment 50 consists of polynucleotide analogs. In oneembodiment, the template segment consists of a chimera of more than onepolynucleotide or polynucleotide analog selected from the groupconsisting of RNA, DNA, polynucleotide analogs of RNA, andpolynucleotide analogs of DNA.

In a set of capture probes 10, the template segments 50 can compriseidentical sequences, different sequences or different in both sequenceand length. For example, template segments 50 comprising polynucleotidesof different lengths in a set of capture probes 10 can be used toproduce different extension products of their respective target smallpolynucleotides such as miRNAs. Further, extension products of differentlengths can then be utilized to distinguish different target small RNAsfrom one another using standard methods, such as for example usingcapillary electrophoresis.

The synthesis of the capture probes 10 entails known methods as will beunderstood by those with skill in the art with reference to thisdisclosure. For example, the method can comprise, first, selecting thesequences of solid phase binding segment 20, the spacer segment 30, thesmall polynucleotide binding segment 40 and the template segment 50, andthen synthesizing them. For example, in one embodiment, the 3′ solidphase binding segment 20 comprises biotin, the spacer segment 30comprises a short DNA polynucleotide segment of 5 nucleotides such asAGCTC, or a polynucleotide such as the T7 DNA dependent RNA promoter,the polynucleotide TAATACGACTCACTATAGGG (SEQ ID NO:9) or itscomplementary sequence CCCTATAGTGAGTCGTATTA (SEQ ID NO:10) or otherpolynucleotide that is not complementary to the small polynucleotide ofinterest 60, the small polynucleotide binding segment 40 comprises oneor more complementary DNA sequence to the small RNA of interest 60, suchas those listed in Table I, and the template segment 50 comprises a DNApolynucleotide sequence such as for example an SP6 DNA dependent RNApolymerase promoter 54, for example the DNA polynucleotideATTTAGGTGACACTATAG (SEQ ID NO:11) or other polynucleotide that is notcomplementary to the small polynucleotide of interest. Additionally, arestriction site 56 can be included in either or both the spacer segment30 and the template segments 50 of the capture probes.

In a particularly preferred embodiment, the penultimate 3′ end of thecapture probe 10 is blocked, for example by phosphate, phosphothioate,biotin, dideoxynucleotide, 3′ amine and the like, so that it cannot beextended. Such blocking of 3′ ends to prevent extension is well known inthe art. The purpose of such a blocking terminus is to prevent extensionof the capture probe 10 by pseudo or latent terminal transferaseactivity inherent in several polymerases.

Synthesis of the capture probes 10 can readily be accomplished byphosphoramidite chemistry and can be obtained from a number of sourceswell known in the art, as will be understood by those with skill in theart with reference to this disclosure. Referring now to Table II, thereare shown 8 sample capture probes 10 useful for detecting the small RNAsof interest 60 listed in the left-hand column (all of which are humanmiRNAs as listed in Table I).

TABLE II PROBE NAME SEQUENCE SEQ ID NO: ILLUM-ED V1-7aATTTAGGTGACACTATAGAACTCGAGAACTATACAAC SEQ ID NO: 12CTACTACCTCAGCTAGCCCCTATAGTGAGTCGTATTA ILLUM-ED V1-7eATTTAGGTGACACTATAGAACTCGAGACTATACAACCT SEQ ID NO: 13CCTACCTCAGCTAGCCCCTATAGTGAGTCGTATTA ILLUM-ED V1-106aATTTAGGTGACACTATAGAACTCGAGGCTACCTGCACT SEQ ID NO: 14GTAAGCACTTTGCTAGCCCCTATAGTGAGTCGTATTA ILLUM-ED V1-126*ATTTAGGTGACACTATAGAACTCGAGCGCGTACCAAA SEQ ID NO: 15AGTAATAATGGCTAGCCCCTATAGTGAGTCGTATTA ILLUM-ED V1-135aATTTAGGTGACACTATAGAACTCGAGTCACATAGGAA SEQ ID NO: 16TAAAAAGCCATAGCTAGCCCCTATAGTGAGTCGTATTA ILLUM-ED V1-138ATTTAGGTGACACTATAGAACTCGAGGATTCACAACA SEQ ID NO: 17CCAGCTGCTAGCCCCTATAGTGAGTCGTATTA ILLUM-ED V1-154ATTTAGGTGACACTATAGAACTCGAGCGAAGGCAACA SEQ ID NO: 18CGGATAACCTAGCTAGCCCCTATAGTGAGTCGTATTA ILLUM-ED V1-154*ATTTAGGTGACACTATAGAACTCGAGAATAGGTCAAC SEQ ID NO: 18CGTGTATGATTGCTAGCCCCTATAGTGAGTCGTATTA PROBE ELEMENTS 5′SP6 SENSE, XhoI, uRNA REVCOMPL, 5′-3′ NheI, T7 REVCOMPL, 3′Biotinorientation optional at synthesis

Methods of Use

Isolation/Capture

According to another embodiment of the present invention, there isprovided a method for isolating a miRNA (microRNA) or other smallpolynucleotides of interest from a sample comprising the smallpolynucleotide of interest. According to another embodiment of thepresent invention, there is provided a method for identifying miRNAs orother small polynucleotides. In one embodiment, the method foridentifying miRNAs or other small polynucleotides comprises, first,isolating the small polynucleotides according to the present invention.Referring now to FIGS. 1A-1D, there are shown some of the steps incertain embodiments of the methods. The steps shown are not intended tobe limiting nor are they intended to indicate that each step depicted isessential to the method, but instead are exemplary steps only.

As can be seen in FIG. 1A, the method comprises, first, providing asample comprising a miRNA or other small polynucleotide of interest 60.Samples suitable for analysis by the present method either comprise orpotentially comprise small RNAs and small DNAs. In one embodiment, thesample further comprises one or more than one substance that ischemically related to the miRNA of interest, such as for example, asubstance selected from the group consisting of messenger RNA, transferRNA, ribosomal RNA, siRNA, 5S/5.8S rRNA, genomic DNA and a combinationof the preceding. In one embodiment, the sample further comprises one ormore than one RNA other than miRNA, such as for example, a substanceselected from the group consisting of messenger RNA, transfer RNA,ribosomal RNA, siRNA, 5S/5.8S rRNA and a combination of the preceding.All of the RNA in the sample, regardless of the type of RNA, constitutesthe “total RNA” in the sample.

In one embodiment, suitable samples are obtained from eukaryotic cellsobtained from whole blood, tissue culture, cell cultures, whole tissuessuch as liver, lung, brain, or even whole organisms such as C. elegansor Drosophila. Small polynucleotides can also be isolated from tissuesinfected by some viruses as these microbes produce miRNAs which cansuppress the immune response or modify other host factors to enabletheir persistence and infection by compromising host factors orotherwise divert host resources to their advantage. Also, smallpolynucleotides can occur in bacteria or procaryotes which regulatetheir processes such as biofilm formation and other activities of thebacteria such as pathogenicity. Such specimen sources are well known inthe art.

In one embodiment, the sample is from a eukaryote. In anotherembodiment, the sample is from a primate. In a preferred embodiment, thesample is from a human.

In one embodiment, the sample comprises a tissue or fluid selected fromthe group consisting of blood, brain, heart, intestine, liver, lung,pancreas, muscle, a leaf, a flower, a plant root and a plant stem.

Cell lysates are suitable for use with the capture probes 10, especiallywhen care has been taken to neutralize nucleases which can degrade themiRNAs or small polynucleotides to be examined in the sample or degradethe capture probes 10 contacted with the sample, however, the captureprobes can be rendered resistant to the action of nucleases by theirsynthesis with nuclease resistant backbones such as amides such aspeptide nucleic acids or more commonly phosphothioate modified backbonesduring their synthesis. In another embodiment the sample is a mounted,fixed tissue section, where the fixed small polynucleotides, for examplemiRNAs, in the sample serve as the solid phase binding segment orelement 20 of the capture-extension probes 10.

In one embodiment, the method further comprises isolating the total RNAfrom the sample after providing the sample. In a preferred embodiment,total RNA is isolated from such specimens using methods well known inthe art or using commercial kits widely available from vendors such asQIAgen, Invitrogen, Promega and the like. As will be understood by thosewith skill in the art with reference to this disclosure, when the methodcomprises isolating the total RNA from the sample after providing thesample, the term “sample” means the isolated total RNA for the remainingsteps in the method.

The small polynucleotide of interest 60 has a small polynucleotide ofinterest sequence, and comprises 3′ end and a 5′ end. In one embodiment,the small polynucleotide of interest is a miRNA, which consists ofbetween 18 and 24 RNA residues. In another embodiment, the miRNA ofinterest consists of 18 or 19 or 20 or 21 or 22 or 23 or 24 RNAresidues.

The small polynucleotide of interest 60 is substantially complementaryto, and capable of hybridizing to, a small polynucleotide bindingsegment 40 of a capture probe 10 according to the present invention byWatson-Crick base pairing. In one embodiment, the small polynucleotideis a miRNA of interest listed in a public database. In a preferredembodiment, the public database is a central repository provided by theSanger Institute http://microrna.sanger.ac.uk/sequences/ to which miRNAsequences are submitted for naming and nomenclature assignment, as wellas placement of the sequences in a database for archiving and for onlineretrieval via the world wide web. Generally, the data collected on thesequences of miRNAs by the Sanger Institute include species, source,corresponding genomic sequences and genomic location (chromosomalcoordinates), as well as full length transcription products andsequences for the mature fully processed miRNA (miRNA with a 5′ terminalphosphate group).

In one embodiment, the sample provided comprises a plurality of miRNAsof interest 60, where each of the plurality of miRNAs or other smallpolynucleotides of interest 60 has small polynucleotide of interestsequences that are identical to one another. In one embodiment, thesample provided comprises a plurality of miRNAs of interest 60, where atleast two of the plurality of miRNAs of interest 60 have miRNA ofinterest sequences that are different from one another. In oneembodiment, the sample provided comprises a plurality of miRNAs ofinterest 60 comprising a first miRNA of interest having a first miRNA ofinterest sequence, and a second miRNA of interest having a second miRNAof interest sequence, where the first miRNA of interest sequence isdifferent from the second miRNA of interest sequence. In anotherembodiment, the sample provided comprises a plurality of miRNAs ofinterest 60 comprising a first miRNA of interest having a first miRNA ofinterest sequence, a second miRNA of interest having a second miRNA ofinterest sequence, and a third miRNA of interest having a third miRNA ofinterest sequence, where the first miRNA of interest sequence isdifferent from the second miRNA of interest sequence, where the firstmiRNA of interest sequence is different from the third miRNA of interestsequence, and where second miRNA of interest sequence is different fromthe third miRNA of interest sequence.

Next, the method further comprises providing a capture probe 10. In oneembodiment, the capture probe 10 provided is a capture probe 10according to the present invention. When the capture probe 10 is acapture probe according to the present invention, in all respects, thecapture probe 10 provided has the characteristics and attributes asdisclosed for a capture probe 10 according to the present invention,some of which will be repeated hereafter for clarity. As can be seen inFIG. 1A, the capture probe 10 comprises three segments depicted in FIG.1A from left to right, from the 3′ end of the capture probe 10 to the 5′end of the capture probe: a) a spacer segment 30 having a spacer segmentsequence; b) a small polynucleotide binding segment 40 having apolynucleotide binding segment sequence; and c) a template segment 50having a template segment sequence, and comprising a 3′ end and a 5′end, where the 5′ end of the spacer segment 50 is connected to the 3′end of the polynucleotide binding segment 40, and where the 5′ end ofthe polynucleotide binding segment 40 is connected to the 3′ end of thetemplate segment 50. The specificity of the polynucleotide bindingsegment 40 to a miRNA or other small polynucleotide of interest 60allows the method to be used directly on a sample containing substancesrelated to miRNA or on isolated total RNA without requiring the specificseparation of miRNAs from the sample or from the total RNA, such as forexample by either gel purification or chromatographic purification, asnecessary in prior art methods.

In a particularly preferred embodiment, the penultimate 3′ end of thecapture probe 10 is blocked, for example by phosphate, phosphothioate,biotin, dideoxynucleotide, 3′ amine and the like, so that it cannot beextended. Such blocking of 3′ ends to prevent extension is well known inthe art. The purpose of such a blocking terminus is to prevent extensionof the capture probe 10 by pseudo or latent terminal transferaseactivity inherent in several polymerases.

In one embodiment, a plurality of capture probes 10 are provided as acomposition or mixture comprising two or more capture probes. Themixture includes (a) a first capture probe 10 having a first spacersegment 30, a first small polynucleotide binding segment 40 and a firsttemplate segment 50; and (b) a second capture probe 10 having a secondspacer segment 20, a second small polynucleotide binding segment 40 anda second template segment 50, where the second small polynucleotidebinding segment 40 has a different small polynucleotide binding segmentsequence than the first small polynucleotide binding segment 40 and thesecond template segment 50 has a different template segment sequencethan the first template segment 50. The presence of different smallpolynucleotides bound to the capture probes 10 can thus be correlated toa detectable difference in the associated template segments 50. In apreferred embodiment, the first template segment 50 and the secondtemplate segment 50 differ in length.

Referring now to FIG. 1A, the method then comprises combining thecapture probe 10 and the sample, represented in FIG. 1A by the smallpolynucleotide of interest 60. In a preferred embodiment, the methodcomprises combining the sample and the capture probe 10 in a solution.

In one embodiment, combining the capture probe 10 and the samplecomprises combining approximately equimolar amounts of each captureprobe 10. In another embodiment, combining the capture probe 10 and thesample comprises combining approximately equimolar amounts of eachcapture probe 10 with an amount of sample expected to containapproximately one tenth the molar amount of the small polynucleotide ofinterest 60 as of the capture probe 10. In another embodiment, combiningthe capture probe 10 and the sample comprises combining approximatelyequimolar amounts of each capture probe 10 with an amount of sampleexpected to contain approximately one half and one tenths and the molaramount of the small polynucleotide of interest as of the capture probe10. In one embodiment, combining the capture probe 10 and the samplecomprises combining the sample with between 0.1 pmoles and 100 pmoles/μleach of the capture probe 10 in a suitable buffer to create a solutioncomprising the capture probe 10 and the sample. In a preferredembodiment the amount of total RNA in the sample ranges from about 10 pgto about 10 μg, more preferably about 10 ng to about 1 μg. In apreferred embodiment, the buffer is selected from the group consistingof TRIS, MOPS, and SSC; includes alkali salts such as sodium chloride,lithium chloride or sodium citrate; and may further include nucleaseinhibitors and accelerants such as dextran sulfate, polyethylene glycolsor polyacrylamides. Exemplary buffers include, (a) 1×TE buffer in0.1-2.0 M sodium chloride; (b) 0.1M MOPS in 1 mM EDTA and 100 mM sodiumchloride, and (c) 20 mM MOPS, 1.8M Lithium Chloride, 1 mM EDTA, 100 μMaurintricarboxylic acid pH 6.8. As will be understood by those withskill in the art with reference to this disclosure, the pH selected forthe buffer will be one that optimizes the intended reactions. Ingeneral, the pH selected will be between 6 and 8, preferably between 6.4and 7.4 and more preferably, near 7.0. In a preferred embodiment, themethod further comprises adding one or more than one RNase inhibitor tothe combination of the sample and the capture probe 10 such as forexample an RNase or nuclease inhibitor selected from the groupconsisting of lithium dodecylsulfate (LiDS), sodium dodecylsulfate, theammonium salt of aurintricarboxylic acid and sodium salt ofaurintricarboxylic acid, beta mercaptoethanol, dithiothreitol,Tris(2-Carboxyethyl)-Phosphine Hydrochloride (TCEP) or human placentalRNase inhibitor. Such inhibitors are included to inhibit nucleaseswithout compromising the ability of the probes and their targetpolynucleotides to hybridize with one another as will be understood bythose skilled in the art.

Referring now to FIG. 1B, after combining the capture probe 10 and thesample, the method comprises allowing the small polynucleotide ofinterest 60 to hybridize with the small polynucleotide binding segment40 to form a small polynucleotide/capture probe complex (FIG. 1B). Inone embodiment, allowing the small polynucleotide of interest 60 tohybridize with the small polynucleotide binding segment 40 comprisesincubating the solution comprising the capture probe 10 and the samplefor between 1 minute and 60 minutes at between 25° C. and 60° C. untilsubstantially all of the miRNA of interest 60 has hybridized to thecapture probes 10, thereby sequestering the small polynucleotide ofinterest 10 from other substances in the sample.

In addition to the small polynucleotide binding segment 40, the captureprobes 10 also contain a solid phase binding segment 20, a spacersegment 30 and a template segment 50 capable of serving as a templatefor a polynucleotide polymerase. The set of capture probes 10 andhybridized target RNAs 60 are then captured to a solid phase, forexample by binding of biotinylated capture probes 10 to streptavidincoated paramagnetic particles followed by temporary immobilization ofthe paramagnetic particles by the action of a magnet and removal of theremaining biological sample. Unlike other methods for determining smallpolynucleotides such as miRNAs, using the method of this disclosurepermits the recovery and further processing of the removed biologicalsample to be analyzed for other molecular species such as mRNAs orgenomic DNA This is followed by cycles of washing the particles aftertheir release into a wash buffer to remove unhybridized polynucleotidesand other materials from the paramagnetic beads and thecapture-extension probe hybridization complexes.

One advantage for the immobilized capture probe 10 methods is thatinitial enrichment of the total RNA sample for non-protein-coding RNAs,such as small nuclear RNAs, siRNAs, microRNAs and antisense RNAs, is notnecessary. Preferably, the capture probe 10 will hybridize to thespecific target in solution. Secondly, when the capture probe 10 isimmobilized on the solid support, unbound material can be removed andthereby enrichment for the specific target has been performed. Anotheradvantage is that buffer exchange can be facilitated. Yet anotheradvantage is that at this point the small polynucleotides can be elutedfrom the bound capture probes. The eluted small polynucleotides arehighly concentrated and enriched and are suitable for use in a widevariety of downstream analytical methods, such elution methods beingwell understood in the art for example use of water or formamide at 80°C., such downstream applications as gel electrophoresis, ligation andsequencing, labeling and hybridization and the like.

Extension

Next, as shown in FIGS. 1B through 1C, the method comprises an extensionreaction. The first step of the extension reaction comprises combiningthe small polynucleotide/capture probe complex with a polynucleotidepolymerase and a set of nucleotide triphosphates. The extension reactionfurther comprises extending the hybridized small polynucleotide ofinterest 60 to form an extension product 80, where the extension product80 is hybridized to the capture probe 10 to form an extensionproduct/capture probe complex (FIG. 1C). The extension product iscomprised of the small polynucleotide of interest 60 connected at the 3′end to an extended segment 70 comprising a sequence complementary to thetemplate segment 50 of the capture probe 10 (FIG. 1D). In one embodimentof the invention, the extended segment contains one or more labeled ormodified nucleotide residues.

Typically, the nucleotide polymerization comprises a DNA polymerizationto obtain a RNA-DNA chimera, which constitutes the extension product 80.In one embodiment of the invention, the hybridized small polynucleotides60 bound to the capture probes 10 are extended by the action ofpolymerase that can utilize the hybridized small RNA as a primer. Inanother embodiment, where the extension template segment 50 of thecapture-extension probe 10 is DNA, the polymerase is a DNA dependent DNApolymerase capable of using the 3′ end of the hybridized smallpolynucleotide 60 as a primer. In another embodiment, the polymerase isa polynucleotide polymerase that can use RNA as primer such as T4, T7,E. coli Pol I, MMLV reverse transcriptase, Bst polymerase, Phi-29polymerase and the like or a combination of one or more of theseenzymes. In a preferred embodiment, the polynucleotide polymerase lacksany nuclease activity and can readily utilize labeled nucleotidetriphosphates as substrates for its extension of the hybridized smallpolynucleotide, such as miRNA which serves as a primer for the extensionreaction.

The nucleotide mixture for the extension reaction is usually a set ofnucleotide triphosphates, usually NTPs, e.g. ATP, CTP, GTP and UTP, ordNTPs, e.g., dATP, dCTP, dGTP and TTP (or dUTP). In one embodiment, atleast one of the nucleotide triphosphates contains a detectable labelsuch as fluorescein, cyanine 3, cyanine 5 biotin, aminoallyl,Digoxigenin, Tetramethyl Rhodamine and the like. A wide variety ofdetectable nucleotide triphosphates are available commercially fromRoche (Indianapolis, Ind., US), Invitrogen (Carlsbad, Calif., US) andothers. In a preferred embodiment, the labeled nucleotide triphosphateis at a lower concentration than the other three nucleotidetriphosphates. For example, in one embodiment, the unlabeled nucleotidetriphosphates are at a concentration in the extension reaction atbetween 50 and 300 micromolar and the labeled nucleotide triphosphatesare at a concentration of between 5 and 30 micromolar. However, as willbe understood in the art, different polymerases have differentcapacities to utilize such modified nucleotides in strand synthesis.Accordingly, in some cases the labeled nucleotide triphosphate may beutilized at concentrations comparable to the non-labeled nucleotidetriphosphates employed in the extension reaction. Such adjustments innucleotide triphosphate concentrations are well known in the art.Additionally, it is known in the art that the buffers and ortemperatures utilized in the extension reaction can be adjusted toaccommodate the incorporation of modified nucleotide triphosphates inthe extension reaction.

The buffer selected for the extension reaction should not interfere withthe hybridization of the small polynucleotide 60 with its capture probe10 and be compatible with the extension reaction caused by thepolymerase. Preferred versions of the buffer permit or facilitate theincorporation of modified nucleotides into the extension product.

In one embodiment the polymerase is a nuclease free form of the Klenowenzyme from E. coli, the nucleotide triphosphates are dATP, dCTP, dGTPat 100 micromolar each, the labeled dNTP is dUTP labeled with cyanine 3at a concentration of 10 micromolar, and the extension buffer comprises0.05M Tris-HCL, 0.01M MgCl₂, 1.0 mM DTT, 0.05 mg/ml BSA and 20 units ofan RNase inhibitor such as a recombinant mammalian protein capable ofinhibiting eukaryotic RNases.

Detection/Identification

Analysis of extension products 80 can be performed using techniquesknown in the art including, without limitation, hybridization anddetection by the use of a microarray specific for the miRNAs or othersmall polynucleotides to be evaluated, polymerase chain reaction(PCR)-based analysis, sequence analysis, flow cytometry andelectrophoretic analysis.

It will also be understood by those skilled in the art that the set ofcapture probes 10 could be initially bound to a solid phase such asfluorescently coded beads with coded beads assigned to identify eachcapture-probe according to its specificity or complementarity to a givensmall polynucleotide. In one embodiment each uniquely coded bead in theset of coded beads corresponds to a unique miRNA within the set ofmiRNAs to be evaluated. Such coded beads for assay by flow methods areavailable from a number of vendors such as Luminex, (Austin, Tex., US).It will be understood by those skilled in the art that the set of codedbeads is then contacted with the biological sample containing the miRNAsto be evaluated or measured under suitable conditions for hybridization.Further, the hybridized beads are then subjected to washing to removenonhybridized materials and other components present in the sample. Thebeads are then extended in the presence of labeled dNTP(s) by the actionof a DNA polymerase as set forth above. It is possible to then directlyassay the plurality of coded beads for the presence and quantity ofmiRNAs or other small polynucleotides in the biological sample. Toreduce background and noise it may be desirable to remove the unboundcomponents of the extension reaction from the beads by washing them andthen performing the analysis by flow detection, such methods being wellunderstood in the art.

Reverse Transcription

Subsequent amplification, detection, and/or identification of thepolynucleotide of interest 60 in many embodiments may further comprisereverse transcription of the resulting extension product 80 to producecDNA. The design of suitable reverse transcription primers and use ofreverse transcriptase to produce cDNA copies of extension products canbe accomplished by any means known to one of skill in the art withreference to the present disclosure.

Amplification

The terms “PCR reaction”, “PCR amplification”, “PCR”, “pre-PCR” and“real-time quantitative PCR” are interchangeable terms used to signifyuse of a nucleic acid amplification system, which multiplies the targetnucleic acids being detected. Examples of such systems include thepolymerase chain reaction (PCR) system and the ligase chain reaction(LCR) system. Other methods recently described and known to the personof skill in the art are the nucleic acid sequence based amplification(NASBATM, Cangene, Mississauga, Ontario, Canada) and Q Beta Replicasesystems. The products formed by said amplification reaction may or maynot be monitored in real time or only after the reaction as an end pointmeasurement.

RNA Amplification

In one embodiment, shown in FIGS. 2A through 2D, the method furthercomprises converting a partial RNA polymerase recognition sequence 54contained within the template segment 50 or spacer segment 30 of thecapture probe into a complete RNA polymerase recognition sequence 54 and72 and ultimately into a double stranded RNA polymerase promoter 54 and72. Subsequent RNA transcription using an RNA polymerase that recognizesthe double stranded RNA polymerase promoter 54 and 72 results in theproduction of amplified single stranded RNA molecules. Such singlestranded RNA molecules 90 find utility in various downstreamapplications, including gene expression studies involving nucleic acidmicroarrays and knockout of corresponding miRNA or other RNAcomplementary to the transcript by antisense or RNAi activity withincells.

The term “RNA polymerase recognition sequence” is intended to cover bothsingle stranded and double stranded nucleotide sequences 54 and 72. Whenin single stranded form, the nucleotide sequence corresponds to thetemplate or non-template strand of a double-stranded RNA polymerasepromoter. “Template strand” refers to a strand of nucleic acid on whicha complementary copy is synthesized from nucleotides or nucleotideanalogs through the activity of a template-dependent nucleic acidpolymerase. “Non-template strand” refers to the nucleic acid strand thatis complementary to the template strand. When in double stranded form,the nucleotide sequences correspond to both the template andnon-template strands of a double-stranded RNA polymerase promoter.

In one embodiment the template segment of the capture probe contains thenon-template strand of a RNA polymerase recognition site 70. In anotherembodiment the spacer segment contains the template strand of a RNApolymerase recognition site.

Any RNA polymerase recognition sequence 54 can be used in the methodsdescribed herein, so long as it is specifically recognized by an RNApolymerase. Preferably, the RNA polymerase recognition sequence used isrecognized by a bacteriophage RNA polymerase, such as T7, T3, or SP6 RNApolymerase. An exemplary T7 RNA polymerase recognition sequence isTAATACGACTCACTATAGGG (SEQ ID NO: 20). An exemplary T3 RNA polymeraserecognition sequence is AATTAACCCTCACTAAAGGG (SEQ ID NO: 21). Anexemplary SP6 RNA polymerase recognition sequence isAATTTAAGGTGACACTATAGAA (SEQ ID NO: 22).

For example, with reference to FIG. 2B, a small polynucleotide ofinterest 60 (e.g., mRNA, hnRNA, rRNA, tRNA, miRNA, siRNA, snoRNA,non-coding RNAs, antisense DNAs, etc.) hybridizes to the smallpolynucleotide binding segment 40 of a capture probe 10, wherein thetemplate segment 50 contains the required RNA polymerase recognitionsite 54 (in the case of FIG. 2B, the non-template strand of the RNApolymerase promoter).

The subsequent extension product 80 comprises the polynucleotide ofinterest 60 and an extended segment 70 adjacent to the 3′ end of thesmall polynucleotide of interest containing a sequence complementary tothe to the RNA polymerase recognition site of the template segment 50(FIG. 2C). The double stranded region comprising the RNA polymeraserecognition site 54 and its complementary sequence 72 generates an RNApolymerase promoter.

In one embodiment, in order to detect a polynucleotide of interest 60,the extension product 80 formed as described above is transcribed usingan RNA polymerase which recognizes the RNA polymerase promoter 54 and 72located at the opposite end of the extension product 80, such that anRNA product 90 is formed comprising a sequence 40 complementary to thepolynucleotide of interest. Combining the double stranded extensionproduct/capture probe complex with an RNA polymerase, which recognizesthe RNA polymerase promoter 54 and 72, produces a single stranded RNAproduct containing sequences complementary to the small polynucleotideof interest., i.e., a cRNA 40 (FIG. 2D). In one aspect, the spacersegment 30 of the capture probe 10 can include an RNA polymerase stopsite 36, for example the T7 stop sequence GCTAGTTATTGCTCAGCGG (SEQ IDNO:23). In this case the cRNA transcripts 90 will contain residuesimmediately adjacent to and downstream from the RNA polymeraserecognition site 54 terminating at the 3′ end at the residue precedingthe stop site 36. If the stop site 36 is omitted then the enzyme willpolymerize the entire sequence downstream of the start of transcriptionin the promoter motif, including the complement of the polynucleotide ofinterest 40 and any other sequence appended to the 5′ end of thepolynucleotide of interest 60, e.g., by ligation of an linker segment.

Preferably the transcription reaction occurs in the presence ofribonucleotides, including labeled ribonucleotides. In one aspect, thenucleotides are labeled. If it is desired to prepare a labeledpolynucleotide comprising cRNA, unlabeled UTP can be omitted andreplaced with or mixed with labeled UTP. Labels can include, forexample, fluorescent labels or radiolabels.

Detection of the RNA transcription product 90 is indicative of thepresence of the suspected polynucleotide of interest 60 in the sampleand can be further used for quantitation of the polynucleotide ofinterest 60. The detection of the transcribed product 90 described abovecan be accomplished by any means known to one of skill in the art.Preferably, the detection is accomplished using detection of a labelincorporated into the transcript 90. Preferably, the detection isperformed after or concurrently with size separation of thetranscription products.

Ligation

Another means to expedite amplification and/or detection of a smallpolynucleotide of interest is to include a ligation reaction to furtherlengthen the small polynucleotide extension product 80. “Ligation” or“covalent coupling” refers to covalent coupling of two adjacentnucleotide sequences, e.g. a linker sequence 100 substantiallycomplementary to, and hybridized to, the spacer segment 30 of thecapture probe covalently coupled to an adjacent miRNA or other smallpolynucleotide extension product 80. The reaction is catalyzed by theenzyme ligase, which forms a phosphodiester bond between the 5′-end ofone nucleotide sequence and the 3′-end of the adjacent nucleotidesequence, e.g. between two adjacent segments of the capture probe orcomplements thereof. Suitable enzymes include the following Ligases: EC6.5.1.1 (DNA ligase (ATP)) and EC 6.5.1.3 (RNA ligase (ATP)).

Following hybridization of the small polynucleotide of interest 60 tothe capture probe 10, the method in accordance with this aspect of thepresent invention further comprises providing a linker segment 100 (FIG.3A). In one embodiment, the linker segment 100 comprises a substanceselected from the group consisting of one or more than one type ofpolynucleotide, including ribonucleotides and deoxynucleotides, one ormore than one type of polynucleotide analog, and a combination of one ormore than one type of polynucleotide and polynucleotide analog. In oneembodiment, the linker 100 is resistant to nuclease degradation. In apreferred embodiment, the linker 100 comprises nuclease resistantnucleotides. In another preferred embodiment, the linker 100 comprisesnucleotides with a phosphothioate backbone that renders the linkerresistant to nuclease degradation.

The linker 100 has a linker sequence, and comprises a 3′ end and a 5′end. In one embodiment, the linker sequence is substantiallycomplementary to, and capable of hybridizing to, the spacer segmentsequence 30 of a capture probe 10 according to the present invention byWatson-Crick base pairing.

The linker 100 comprises between 6 and 50 residues. In a preferredembodiment, the linker 100 comprises at least 10 residues, and at least10 residues at the 3′ end of the linker 100 are exactly the complementof the corresponding residues at or near the 5′ end of the spacersegment 30.

In one embodiment, the linker 100 is allowed to hybridize to the spacersegment 30 and is then ligated to the extension product 80 to form aligated extension product 110 substantially complementary to, andcapable of hybridizing to, the capture probe sequence 10 (FIG. 3B). Suchligation reaction may be assisted by providing a linker 100 havinglinker sequence specific for the spacer segment sequence 30 of thecapture probe 10 so that the small polynucleotide target 60 and saidlinker 100 are placed in close vicinity to each other upon sequencespecific hybridization.

In a preferred embodiment, the 3′ end of the linker 100 is capable ofbeing ligated to the 5′ end of a miRNA of interest 60 by a suitableligase, such as for example T4 polynucleotide ligase, or by anothersuitable chemical reaction.

Referring now to FIG. 3A, the method then comprises combining the linker100 with the sample and the capture probe 10, represented in FIG. 3A bythe small polynucleotide of interest 60 and the extended segment 70hybridized to the capture probe 10. In a preferred embodiment, themethod comprises combining the linker 100 and the hybridized captureprobe/extension product 10/80 in a solution. Alternatively, the captureprobe 10, the linker 100 and the sample can be combined simultaneously,or sequentially, in any order, as will be understood by those with skillin the art with reference to this disclosure. For example, the captureprobe 10 is combined with the sample first, and then the capture probe10 and sample are combined with the linker 100; or alternately forexample, the capture probe 10 and linker 100 are combined first, andthen the capture probe 10 and linker 100 are combined with the sample;or alternately for example, the linker 100 is combined with the samplefirst, and then the capture probe 10 is combined with the linker 100 andthe sample.

In one embodiment, combining the capture probe 10, the linker 100 andthe sample comprises combining approximately equimolar amounts of thecapture probe 10 and the linker 100. In another embodiment, combiningthe capture probe 10, the linker 100 and the sample comprises combiningapproximately equimolar amounts of the capture probe 10 and the linker100 with an amount of sample expected to contain approximately one tenththe molar amount of small polynucleotide of interest 60 as of thecapture probe 10 or linker 100. In one embodiment, combining the captureprobe 10, the linker 100 and the sample comprises combining the samplewith between 0.1 pmoles and 100 pmoles/μl each of the capture probe 10and the linker 100 in a suitable buffer to create a solution comprisingthe capture probe 10, the linker 100 and the sample. In a preferredembodiment, the buffer is selected from the group consisting of TRIS,MOPS, and SSC; includes alkali salts such as sodium chloride, lithiumchloride, sodium citrate; and may further include nuclease inhibitorsand accelerants such as dextran sulfate, polyethylene glycols,polyacrylamides, Exemplary buffers include, (a) 1×TE buffer in 0.1-2.0 Msodium chloride; (b) 0.1M MOPS in 1 mM EDTA and 100 mM sodium chloride,and (c) 20 mM MOPS, 1.8M Lithium Chloride, 1 mM EDTA, 100 μMaurintricarboxylic acid pH 6.8. As will be understood by those withskill in the art with reference to this disclosure, the pH selected forthe buffer will be one that optimizes the intended reactions. Ingeneral, the pH selected will be between 6 and 8, preferably between 6.4and 7.4 and more preferably, near 7.0. In a preferred embodiment, themethod further comprises adding one or more than one RNase inhibitor tothe combination of the sample and the capture probe 10 such as forexample an RNase or nuclease inhibitor selected from the groupconsisting of lithium dodecylsulfate (LiDS), sodium dodecylsulfate, theammonium salt of aurintricarboxylic acid and sodium salt ofaurintricarboxylic acid, beta mercaptoethanol, dithiothreitol,Tris(2-Carboxyethyl)-Phosphine Hydrochloride (TCEP) or human placentalRNase inhibitor. Such inhibitors and included to inhibit nucleaseswithout compromising the ability of the probes and their targetpolynucleotides to hybridize with one another as will be understood bythose skilled in the art.

Referring now to FIG. 3B, after combining the linker 100, the captureprobe 10 and the sample, the method comprises allowing the linker 100 tohybridize with the spacer segment 30, thereby binding the linker 100,the small polynucleotide of interest 60, and optionally the extendedsegment 70 to the capture probe 10. In one embodiment, allowing thelinker 100 to hybridize with the spacer segment 30 and the smallpolynucleotide of interest 60 to hybridize with the small polynucleotidebinding segment 40 comprises incubating the solution comprising linker100, the capture probe 10 and the sample for between 1 minute and 60minutes at between 25° C. and 60° C. under conditions sufficient tohybridize the linker 100 to the spacer segment 30 of the capture probe10.

In a preferred embodiment, the linker 100 hybridizes to the spacersegment 30 at a position where the last residue on the 3′ end of thelinker 100 hybridizes to a residue on the spacer segment 30 that isbetween 1 residue and 5 residues from the 5′ end of the smallpolynucleotide of interest 60. In a particularly preferred embodiment,the linker 100 hybridizes to the spacer segment 30 at a position wherethe last residue on the 3′ end of the linker 100 hybridizes to a residueon the spacer segment 30 that is immediately adjacent to the 5′ end ofthe small polynucleotide of interest 60.

Next, as shown in FIG. 3B, the method comprises covalently ligating the3′ end of the linker 100 that is hybridized to the spacer segment 30 tothe 5′ end of the small polynucleotide of interest 60 that is hybridizedto the small polynucleotide binding segment 40. Ligation of the 3′ endof the linker 100 to the 5′ end of the small polynucleotide of interest60, and extension of the 3′ end of the small polynucleotide of interestto the 3′ end of the extended segment 70 can be accomplished in anyorder, including simultaneously or sequentially. In one embodiment, theligation is accomplished by standard techniques, as will be understoodby those with skill in the art with reference to this disclosure. In apreferred embodiment, the ligation comprises treating the capture probe10 with the hybridized linker 100 and the extension product 80containing a small polynucleotide of interest 60 with a suitable ligase,such as for example T4 polynucleotide ligase in the presence of suitablebuffer and essential cofactors for a sufficient time for the ligation toproceed to near total completion of ligation. As will be understood bythose with skill in the art with reference to this disclosure, thepresence of the spacer segment 30 in the capture probe 10 facilitatesthe ligation of the linker 100 to the small polynucleotide of interest60 by aligning the 3′ end of the linker 100 with the 5′ end of the smallpolynucleotide of interest 60. The combination of ligation and extensionsteps produces a “ligated extension product” 110 defined as a strand oflinker 100, small polynucleotide of interest 60 and extended segment 70that have been covalently linked together (“ligated linker-smallpolynucleotide of interest-extended segment”), and where the ligatedextension product 110 is hybridized to the capture probe 10.

In one embodiment, the 5′ end of the linker 100 comprises a label, suchas for example a fluorescent dye, to facilitate detection, as will beunderstood by those with skill in the art with reference to thisdisclosure. Further, the linker 100 can comprise a label, such as forexample a fluorescent dye, to facilitate detection at a position otherthan at the 5′ end of the linker 100, as long as the presence of thelabel does not interfere with other steps of the present method, as willbe understood by those with skill in the art with reference to thisdisclosure. In other embodiments, the linker sequence 100 joined to thesmall polynucleotide of interest 60 by ligation may accommodate in partprimers for PCR amplification or for a labeled detection probe, alone orin combination with the nucleic acid sequence of the adjacent smallpolynucleotide 60.

Detector Probes

In some embodiments, the detection and identification of thepolynucleotide of interest can employ a detector probe. “Detector probe”refers to an a nucleic acid binding molecule capable of recognizing aparticular target nucleotide sequence, typically used in anamplification reaction, which can include quantitative or real-time PCRanalysis, as well as endpoint analysis. Such detector probes canlikewise be used to monitor the extension, reverse transcription and/oramplification of the target small polynucleotides, appended segmentsand/or complements thereof.

Detector probes typically include a fluorescent molecule or fluorophore,including without limitation sulfonate derivatives of fluorescein dyeswith SO3 instead of the carboxylate group, phosphoramidite forms offluorescein, phosphoramidite forms of CY3 or CY5 (commercially availablefor example from Amersham), aminoallyl, Digoxigenin, TetramethylRhodamine and the like. A wide variety of detectable nucleotidetriphosphates are available commercially from Roche (Indianapolis, Ind.,US), Invitrogen (Carlsbad, Calif., US) and others.

The term “Dual-labeled probe” refers to an oligonucleotide with twoattached labels. In one aspect, one label is attached to the 5′ end ofthe probe molecule, whereas the other label is attached to the 3′ end ofthe molecule. A particular type of dual-labeled probe contains afluorescent molecule attached to one end and a molecule which is able toquench this fluorophore by Fluorescence Resonance Energy Transfer (FRET)attached to the other end. Accordingly, a dual-labeled detector probecan also comprise a quencher, including without limitation Black Holequenchers (Biosearch, Novato, Calif., US), Iowa Black (IDT, Coralville,Iowa, US), QSY quencher (Molecular Probes, Invitrogen, Carlsbad, Calif.,US), and Dabsyl and Dabcyl sulfonate/carboxylate Quenchers (Epoch,Bothell, Wash., US).

“5′ nuclease assay probe” refers to a dual-labeled probe which may behydrolyzed by the 5′-3′ exonuclease activity of a DNA polymerase. Probedegradation allows for the separation of the fluorophore and thequencher, resulting in increased fluorescence emission. “5′ nucleaseassay probes” are often referred to as a “TaqMan assay probes”, and the“5′ nuclease assay” as “TaqMan assay”. These names are usedinterchangeably in this application.

“Molecular Beacon” refers to a single or dual-labeled probe which is notlikely to be affected by the 5′-3′ exonuclease activity of a DNApolymerase. Special modifications to the probe, polymerase or assayconditions have been made to avoid separation of the labels orconstituent nucleotides by the 5′-3′ exonuclease activity of a DNApolymerase. For example, a particular aspect of the molecular beacon maycontain a number of nuclease resistant residues to inhibit hydrolysis bythe 5′-3′ exonuclease activity of a DNA polymerase. The detectionprinciple thus relies on a detectable difference in label elicitedsignal upon binding of the molecular beacon to its target sequence. Inone aspect of the invention the oligonucleotide probe forms anintramolecular hairpin structure at the chosen assay temperaturemediated by complementary sequences at the 5′- and the 3′-end of theoligonucleotide. The oligonucleotide may have a fluorescent moleculeattached to one end and a molecule attached to the other, which is ableto quench the fluorophore when brought into close proximity of eachother in the hairpin structure.

The detection of binding is either direct by a measurable change in theproperties of one or more of the labels following binding to the target(e.g. a molecular beacon type assay with or without stem structure) orindirect by a subsequent reaction following binding, e.g. cleavage bythe 5′ nuclease activity of the DNA polymerase in 5′ nuclease assays. Insome embodiments, a dual-labeled probe having an intramolecular hairpinstructure, such as the stem-loop and duplex Scorpion™ probes, can serveas both a primer and probe during amplification.

Detector probes can also comprise two probes, wherein for example afluorophore is on one probe, and a quencher is on the other probe,wherein hybridization of the two probes together on a target quenchesthe signal, or wherein hybridization on the target alters the signalsignature via a change in fluorescence.

In some embodiments, DNA binding dyes, which emit fluorescence whenbound to double stranded DNA, can be used to detect double stranded DNAproducts, which accumulate during amplification. Non-covalently boundminor groove binders (MGB) and/or intercalating labels are used, such asasymmetric cyanine dyes, DAPI, SYBR Green I, SYBR Green II, SYBR Gold,PicoGreen, thiazole orange, Hoechst 33342, Ethidium Bromide,1-O-(1-pyrenylmethyl)glycerol and Hoechst 33258, thereby allowingvisualization in real-time, or end point, of an amplification product inthe absence of a detector probe. In some embodiments, real-timevisualization can comprise both an intercalating detector probe and asequence-based detector probe can be employed.

In one embodiment, shown in FIG. 4A, the method comprises: (a) providinga sample comprising the small polynucleotide of interest 60, the captureprobe 10, and a dual-labeled detector probe 120, having one labelattached to the 5′ end of detector probe molecule, another labelattached to the 3′ end of the detector probe 120, and a detector probesequence that is substantially complementary to, and capable ofhybridizing to a detector probe binding sequence 52 within the templatesegment 50 of the capture probe 10. After combining the sample, captureprobe 10 and the detector probe 120, as shown in FIG. 4A, the smallpolynucleotide of interest 60 is allowed to hybridize with thepolynucleotide binding segment 40 and the detector probe 120 is allowedto hybridize with the detector probe binding sequence 52 of the captureprobe 10.

In one embodiment the polynucleotide polymerase preferably has a 5′exonuclease activity capable of cleaving polynucleotides downstream ofthe hybridized small polynucleotide 60 during extension. As shown inFIG. 4B, upon adding the polymerase and nucleotide mix to the reactionmixture, the hybridized small polynucleotide of interest 60 is extendedto form the extension product, whereas the detector probe 120 may behydrolyzed by the 5′ to 3′ exonuclease of the polynucleotide polymerase.Alternatively, binding of the small polynucleotide of interest 60 isdetected by measuring the change in fluorescence properties of one ormore of the labels following cleavage of the detector probe 120 andconsequent separation of the two labels.

In another embodiment the extended segment 70 of extension product 80and the detector probe contain complementary sequences recognized by arestriction enzyme. When the detector probe and extended segment arecombined to form a double stranded hybridization complex, the detectorprobe sequence can be recognized and cut by the restriction enzyme. In apreferred embodiment, the restriction enzyme is a nicking endonucleasethat “nicks” a single strand of the complex. Alternatively, theextension reaction can include one or more nucleotide analogs in thereaction mixture that render the extended segment resistant toendonuclease action. Nicking of the probe then provides a detectablechange in fluorescence.

Nicked Extension Products

In one embodiment, shown in FIGS. 5A through 5D, the method comprisesproviding a capture probe, where the template segment 50 comprises oneor more than one sequence that is one strand of a double strandedrestriction enzyme recognition motif 56, referred to herein as arestriction site (FIG. 5A).

Next, as disclosed above, the method comprises combining the captureprobe and the sample, containing the small polynucleotide of interest60, and allowing the small polynucleotide of interest 60 to hybridizewith the small polynucleotide binding segment 40 to form a smallpolynucleotide/capture probe complex. Next, as shown in FIGS. 5A and 5B,the method comprises an extension reaction, where the smallpolynucleotide/capture probe complex is combined with a polynucleotidepolymerase and a set of nucleotide triphosphates. The extension reactionfurther comprises extending the hybridized small polynucleotide ofinterest to form an extension product, where the extension product ishybridized to the capture probe to form an extension product/captureprobe complex. In one aspect, the extension step converts a singlestranded restriction enzyme recognition sequence 56 contained within thetemplate segment 50 of the capture probe into a double strandedrestriction enzyme recognition sequence 56 and 74.

In one embodiment shown in FIG. 5C, the double stranded restriction site56, 74 can be recognized and acted on by a nicking agent. In a preferredembodiment the restriction site motif 74 of the extended segment 70 canbe cut by a nicking endonuclease. In another embodiment, the restrictionenzyme recognition motif 56 of the template segment 50 contains one ormore than one nucleotide analogue, which protects the template segment50 from the endonuclease activity of the nicking agent. For example,precise nicking within a restriction site may be facilitated by makingcertain of its internucleoside bonds resistant to hydrolysis, such as byconverting them to phosphorothioate, boranophosphate, methylphosphonate,or peptide bonds, or by substituting a nucleotide variant that is notrecognized by a specific restriction endonuclease. In one particularaspect, the specificity of a restriction enzyme may preclude recognitionand cutting of a sequence containing dU, substituted for a dT orsimilarly the use of dI (deoxyinosine) substituted for dG, but stillrecognize and act upon the complementary sequence of the oppositestrand.

The next step, shown in FIG. 5C, comprises contacting the extensionproduct/capture probe complex with a nicking agent such that theextension product is selectively nicked on one strand of the doublestranded restriction site 74 to produce a nicked extension product 82.Nicking of the extension product results in a 3′ ended fragmentcontaining the small polynucleotide of interest 60 and a 5′ endedfragment containing a portion of the extended segment 70, referred toherein as the nicked extension fragment 82.

As shown in FIG. 5D, the method further comprises extending the 3′-endedfragment of the nicked extension product containing the hybridized smallpolynucleotide of interest 60, with a polymerase such that the nickingsite is rejuvenated and the nicked extension fragment 82 is displaced.

Displacement of the nicked extension fragment 82 can be facilitated bythermal denaturation and dislodgement of the nicked extension fragment82. Thus, in preferred embodiments the polymerase and restrictionenzymes are thermostable enzymes, which are able to retain enzymaticactivity at elevated temperatures suitable for denaturation anddislodgement of the nicked extension fragments. Potentially applicableDNA polymerases include Vent (exo-), Deep Vent (exo-), Pfu (exo-), Bst(large fragment), Bca (exo-), phi 29, T7 (exo-), and Klenow (exo-).Thermophilic polymerases and/or highly processive polymerases,especially those with strand displacement activity, are likelyadvantageous.

In one embodiment, further cycles of extension, nicking and displacementsteps leads to a linear amplification of nicked extension fragments 82,reflecting the initial concentration of the target small polynucleotide60 originally hybridized to the capture probe.

Detecting the Nicked Extension Products

Detection of the nicked extension fragment 82 is indicative of thepresence of the suspected polynucleotide of interest 60 in the sampleand can be further used for quantitation of the polynucleotide ofinterest 60. The detection of the nicked extension fragment 82 describedabove can be accomplished by any means known to one of skill in the artusing techniques known in the art including, without limitation,detection of a label incorporated into the nicked extension fragment 82,hybridization and detection of one or more than one nicked extensionfragments 82 to a microarray, cloning and sequencing or quantitativeReal-Time PCR.

In one embodiment, detection of a nicked extension fragment 82 can befacilitated by contacting an signal generating probe, having a sequencewhich is complementary to and capable of hybridizing to the displacednicked extension fragment 82. In a preferred embodiment, the signalgenerating probe is about 15 to 25 nucleotide residues in length.Hybridization of the signal generating probe to the nicked extensionfragment 82 to form a double stranded complex can then be detected usingan intercalating dye, such as SYBR green or ethidium bromide, which iscapable of producing a fluorescent signal upon selectively binding todouble stranded nucleic acids. In a preferred embodiment the nickedextension fragment 82 has a nicked extension fragment sequence that canbe correlated with a specific small polynucleotide of interest 60. In apreferred embodiment a first signal generating probe has a first signalgenerating probe sequence and a second signal generating probe has asecond signal generating probe sequence, each having a predeterminedsequence or predetermined size, where either the sequence or the size orboth the sequence and the size of the first signal generating probesequence and the second signal generating probe sequence differ from oneanother. Detecting and distinguishing the sequence and/or size of two ormore signal generating probes permits detection of several targets perassay.

In a further embodiment, the signal generating probe can have afluorophore capable of energy transfer from the intercalating dye. In apreferred embodiment a first signal generating probe has a firstfluorophore and a second signal generating probe has a secondfluorophore, wherein the fluorescence emission spectrum of the firstfluorophore is distinguishable from the fluorescence emission spectrumof the second fluorophore. Detecting and distinguishing the differentfluorescence emission spectra of two or more fluorophores permitsmultiplexing of several targets per assay.

Similarly, in an additional embodiment the signal generating probe whichhybridizes with the displaced nicked extension fragment can be amolecular beacon such that in its unhybridized state its quencher andfluor are proximate to one another via its hairpin structure but whenhybridized with the displaced nicked extension fragment detectablefluorescence is observed because the fluor and quencher are separatedfrom one another and fluorescence is permitted. Consequently thefluorescence will be proportional to the original small polynucleotidepresent in the sample. Molecular beacons can be designed to hybridize tounique nicked extension fragments which can be correlated to thetargeted small polynucleotide by each uniquely targeted capture probe asdiscussed above.

In one embodiment, shown in FIGS. 6A through 6D, the method comprisesproviding a capture probe, where the template segment comprises a firstrestriction site 56 and a second restriction site 58, where the firstrestriction site 56 serves as a template to generate a nick site in theextension product 80 or top strand, and the second restriction site 58is modified to resist nicking of the template segment 50 or bottomstrand. Examples of capture probes in accordance with this aspect of thepresent invention are shown in Table III.

Next, as disclosed above, the method comprises combining the captureprobe and the sample, containing the small polynucleotide of interest,and allowing the small polynucleotide of interest 60 to hybridize withthe small polynucleotide binding segment 40 to form a smallpolynucleotide/capture probe complex. Next, the method comprises anextension reaction, where the small polynucleotide/capture probe complexis combined with a polynucleotide polymerase and a set of nucleotidetriphosphates. The extension reaction further comprises extending thehybridized small polynucleotide of interest 60 to form an extensionproduct, where the extension product is hybridized to the capture probeto form an extension product/capture probe complex. In one aspect, theextension step converts the first restriction site 56 of the captureprobe into a double stranded restriction enzyme recognition sequencecapable of being nicked on the top strand 74, i.e., the extended segment70, but not the bottom strand 56, i.e. template segment 50, andconversely the second restriction site 58 in the capture probe isconverted into a second double stranded restriction recognition sequence58, 76, which is not selectively nicked on the corresponding templatesegment 50.

The next step, shown in FIG. 6A, comprises contacting the extensionproduct/capture probe complex with a nicking agent which recognizes andacts on the first restriction site 56, 74, such that the extensionproduct is selectively nicked on one strand, i.e., the top strand 74, ofthe double stranded restriction site 56, 74 to produce a nickedextension fragment. In one embodiment the nicking agent is a nickingendonuclease and the restriction site motif 74 of the extended segment70 is cut by the nicking agent. In another embodiment, the restrictionenzyme recognition motif of the template segment 50 contains one or morethan one nucleotide analogue, which protects the template segment 50from the endonuclease activity of the nicking agent. For example,precise nicking within a restriction site may be facilitated by makingcertain of its internucleoside bonds resistant to hydrolysis, such as byconverting them to phosphorothioate, boranophosphate, methylphosphonate,or peptide bonds, or by substituting a nucleotide variant that is notrecognized by a specific restriction endonuclease. In one particularaspect, the specificity of a restriction enzyme may preclude recognitionand cutting of a sequence containing dU, substituted for a dT, but stillrecognize and act upon the complementary sequence of the oppositestrand. Examples of capture probes containing a first Nb.BbvCI orNt.BbvCI restriction site and a second Nt.AlwI restriction site, wherein the second site contains a deoxy-U substituted for deoxy-T are shownin Table III below. Additional examples of capture probes containing afirst Nb.BbvCI or Nt.BbvCI restriction site and a second Nt.AlwIrestriction site, where in the second site contains a thiolated backboneare also shown in Table V below.

As shown in FIG. 6B, nicking of the extension product results in a 3′ended fragment containing the small polynucleotide of interest 60 and a5′ ended fragment containing a portion of the extended segment 70,referred to herein as the nicked extension fragment 82.

The method further comprises displacement of the nicked extensionfragment 82, which contains an unmodified top strand of the secondrestriction site 76, by the action of the polymerase. In one embodiment,shown in FIG. 6C, detection of a nicked extension segment 82 can befacilitated by contacting a detector probe 120, which is complementaryto and capable of hybridizing to the nicked extension fragment 82.Formation of the double stranded probe/nicked extension segment complexregenerates the second double stranded restriction recognition sequence,which is now capable of being nicked on the bottom strand, i.e. theprobe sequence 120. In a preferred embodiment, the detection probe isabout 15 to 25 nucleotide residues in length.

Next, the method further comprises contacting the double strandedprobe/nicked extension segment complex with a nicking agent capable ofrecognizing and nicking the detection probe sequence 120. In a preferredembodiment the probe is a dual labeled detector probe 120 so that thenicking reaction provides a detectable change in fluorescence. Examplesof dual label detector probe sequences in accordance with thisparticular embodiment are shown in Table IV.

TABLE III MIRNA CAPTURE PROBES WITH TWO NICK SITES PROBE NAMESEQUENCE 5′-3′ SEQ ID NO: 34 AMP dU_HSA¹-miR-135aATCAGGA/ideoxyU²/CAGCTGAGCCTCAGCATTC SEQ ID NO: 24ACATAGGAATAAAAAGCCATAACAC 38 AMP dU_HSA¹-miR-138ATCAGGA/ideoxyU²/CAGCTGAGCCTCAGCATGA SEQ ID NO: 25 TTCACAACACCAGCTACAC56 AMP dU_HSA¹-miR-154 ATCAGGA/ideoxyU²/CAGCTGAGCCTCAGCATCGSEQ ID NO: 26 AAGGCAACACGGATAACCTAACAC 34 AMP dU_HSA¹-miR-135aATCAGGATCAG*C*³TGAGCCTCAGCATTCACA SEQ ID NO: 27 TAGGAATAAAAAGCCATAACAC38 AMP dU_HSA¹-miR-138 ATCAGGATCAG*C*³TGAGCCTCAGCATGATTCACASEQ ID NO: 28 ACACCAGCTACAC 56 AMP dU_HSA¹-miR-154ATCAGGATCAG*C*³TGAGCCTCAGCATCGAAGGCA SEQ ID NO: 29 CACGGATAACCTAACAC¹“HSA” in the names corresponds to the respective Human miRNA bound by agiven capture probe ²“ideoxyU” corresponds to a dU substitution for T³“*” corresponds to phosphorothioate linkages in the backbone

TABLE IV DUAL LABEL DETECTION PROBES NAME SEQUENCE 5′-3′ SEQ ID NO:QF Probe 1 5′_3′ /5IAbFQ/CAGGATCAGCTGAGAGCC/IFluorT/CA/3Phos/SEQ ID NO: 30 QF Probe 2 5′_3′ /5IAbFQ/CAGGATCAGCTGAGAGCCTCA/36-FAM/SEQ ID NO: 31 “5IAbFQ” corresponds to 5′ Iowa Black FQTM IDT) “IFluorT”corresponds to a fluoroscein labeled T “36-FAM” corresponds to a 3′terminal fluorescein

Incorporation of DNAzyme Motif

In one embodiment, shown in FIGS. 7A through 7C, the method comprisesproviding a capture probe, where the template segment 50 comprises oneor more than one sequence that is complementary to a RNA-cleavingcatalytic nucleic acid, referred to herein as a “DNAzyme motif” 130.Examples of a RNA-cleaving DNA enzyme (DNAzyme) include the “10-23” andthe “8-17” general purpose RNA-cleaving DNA enzymes, which both containconserved catalytic sequences GGCTAGCTACAACGA (SEQ ID NO:32) andTCCGAGCCGGACGA (SEQ ID NO:33), respectively. The conserved catalyticdomain is flanked by variable binding domains capable of hybridizing toa target RNA by Watson-Crick base pairing. Hybridization of the flankingbinding domains to a target RNA results in a loop structure containingthe catalytic domain. Cleavage by an exemplary “10-23” DNAzyme occurs ata purine-pyrimidine dinucleotide of the target RNA, whereas cleavage byan exemplary “8-17” DNAzyme can occur at an AG dinucleotide of thetarget RNA. Accordingly, a DNAzyme complement 130 in accordance with thepresent embodiment will contain sequences complementary to a conservedcatalytic sequence, as will be understood by one of skill in the artwith reference to the present disclosure. In addition to a DNAzymecomplement motif 130, the template segment 50 in accordance with thepresent embodiment, comprises a first flanking segment 132 and a secondflanking segment 134, flanking the 5′ end and the 3′ end of the DNAzymecomplement, respectively. Preferably, the first flanking segment andsecond flanking segment are each about five to twenty nucleotideresidues in length, more preferably about seven to ten, and mostpreferably about eight to nine.

In one embodiment, the template segment 50 further comprises arestriction site 56, as described above. In a preferred embodiment, therestriction site 56 is located downstream from, i.e. in the 3′direction, and may overlap with the second probe segment.

Table V provides examples of capture probes containing a DNAzymecomplement complementary to the “10-23” conserved catalytic sequences,where the DNAzyme complement is flanked on the 5′ end by a nine residuescomprising first flanking segment and on the 3′ end by nine residuescomprising the second flanking segment. In addition the capture probesof Table V all contain a restriction enzyme recognition motif that canbe recognized by the nicking endonuclease N.BbvCI (New England Biolabs,Ipswich Mass., US).

Next, as disclosed above, the method comprises combining the captureprobe and the sample, containing the small polynucleotide of interest60, and allowing the small polynucleotide of interest 60 to hybridizewith the small polynucleotide binding segment 40 to form a smallpolynucleotide/capture probe complex. Next, the method comprises anextension reaction, where the small polynucleotide/capture probe complexis combined with a polynucleotide polymerase and a set of nucleotidetriphosphates. The extension reaction further comprises extending thehybridized small polynucleotide of interest 60 to form an extensionproduct, where the extension product is hybridized to the capture probeto form an extension product/capture probe complex.

In a preferred embodiment, shown in FIG. 7A, the next step comprisescontacting the extension product/capture probe complex with a nickingagent which recognizes and acts on the restriction site 74, such thatthe extension product is selectively nicked on one strand, i.e., the topstrand 74, of the double stranded restriction site to produce a nickedextension fragment 82.

In one embodiment, shown in FIGS. 7B and 7C, displacement of theextension product, or preferably a nicked extension fragment 82,provides a functional DNAzyme capable of hybridizing to and cleaving asuitable substrate. A suitable substrate probe 140 can be an RNApolynucleotide or a chimeric RNA/DNA polynucleotide. The substrate probe140 comprises a first probe segment 132 having a first probe sequence, acleavage site and a second probe segment 134 having a second probesequence. The first probe sequence of the substrate probe 140 issubstantially identical to the first flanking sequence 132 of thetemplate segment 50. Likewise, the second probe sequence 134 of thesubstrate probe 140 is substantially identical to the second flankingsequence 134 of the template segment 50. In a preferred embodiment, theDNAzyme complement motif 78 contains sequence complementary to the“10-23” conserved catalytic domain.

A substrate probe 140 can be a RNA polynucleotide or a chimeric DNA/RNApolynucleotide, having a DNAzyme sensitive cleavage site 142 comprisingone or more than one ribonucleotide residue. The substrate probe 140comprises a first probe segment 132 having a first probe sequence, thecleavage site 142 and a second probe segment 134 having a second probesequence. The first probe sequence 132 of the substrate probe 140 issubstantially identical to the first flanking sequence 132 of thetemplate segment 50. Likewise, the second probe sequence 134 of thesubstrate probe 140 is substantially identical to the second flankingsequence 134 of the template segment 50. In a preferred embodiment thecleavage site 142 comprises a purine residue adjacent to a pyrimidineresidue.

In one embodiment, a loop structure is formed in the nicked extensionfragment 82 by Watson-Crick base pairing between the probe sequences132, 134 and the complementary sequences 136, 138 contained within theextension product 80 or nicked extension fragment 82. Next, the methodfurther comprises nicking the substrate probe at the cleavage site 142.In a preferred embodiment the substrate probe 140 is a dual labeleddetector probe so that the nicking step provides a detectable change influorescence. Examples of dual labeled substrate probes in accordancewith this particular embodiment are shown in Table VI.

TABLE V CAPTURE PROBES CONTAINING DNAZYME MOTIFS NAME SEQUENCE 5′-3′SEQ. ID NO. S1 DNZ 34hsa-miR-135a CAGGACGACTCGTTGTAGCTAGCCTGTACCTCASEQ ID NO: 34 GCATATTCACATAGGAATAAAAAGCCATAAC S1 DNZ 38hsa-miR-138CAGGACGACTCGTTGTAGCTAGCCTGTACCTCA SEQ ID NO: 35GCATATGATTCACAACACCAGCTAC S1 DNZ 56hsa-miR-154CAGGACGACTCGTTGTAGCTAGCCTGTACCTCA SEQ ID NO: 36GCATATCGAAGGCAACACGGATAACCTAAC S2 DNZ 34hsa-miR-135aACATTCACCTCGTTGTAGCTAGCCTTGACCTCA SEQ ID NO: 37GCGAATTCACATAGGAATAAAAAGCCATAAC S2 DNZ 38hsa-miR-138ACATTCACCTCGTTGTAGCTAGCCTTGACCTCA SEQ ID NO: 38GCGAATGATTCACAACACCAGCTAC S2 DNZ 56hsa-miR-154ACATTCACCTCGTTGTAGCTAGCCTTGACCTCA SEQ ID NO: 39GCGAATCGAAGGCAACACGGATAACCTAAC

TABLE VI DUAL LABELED DNAZYME SUBSTRATE PROBES NAME SEQUENCE 5′-3′SEQ ID NO. S1 FAM SUBS ZYME1 /5IABFQ/CAGGACGArCrGrUGTACCTCA/36-fam/SEQ ID NO: 40 S2 CY3 SUBS ZYME2 /5IABFQ/ACATTCACrCrGrUTGACCTCA/3CY3SP/SEQ ID NO: 41

Kits

In another embodiment of the present invention, there is provided a kitcontaining one or reagents for use in the isolation, labeling, anddetection of small RNAs, such as for example, human miRNAs. Preferredversions of the kits can include, (a) an equimolar mix of captureprobes; (b) a nucleotide mix containing deoxyribonucleotidetriphosphates or ribonucleotide triphospates; (c) a polymerase, (d)streptavidin coated paramagnetic beads; (e) one or more than one duallabeled detector probe, (f) a ligase enzyme; (g) an oligonucleotidelinker that is substantially complementary to and capable of hybridizingto the spacer segment of the capture probes; or (h) one or more thanrestriction enzyme specific for a restriction enzyme recognitionsequence contained in the capture probes.

In one embodiment, the kit comprises an equimolar mix of captureextension probes according to the present invention, such as for examplethose listed in Table II, with the small RNA binding segment comprisingcomplementary sequences to the known human mature miRNA population. Inone embodiment, the kit further comprises one or more than one substanceselected from the group consisting of labeling buffer comprising 0.5MTris-HCL, 0.1M MgCl2 10 mM DTT, 0.5 mg/ml BSA and an RNase inhibitor,such as a recombinant mammalian protein capable of inhibiting eukaryoticRNases; a nucleotide mix containing for example Cyanine 3-dUTP orCyanine 5-dUTP at 10 micromolar each and unlabeled dATP, dCTP and dGTPat 100 micromolar each; a labeling enzyme such as a polynucleasepolymerase for example, Exonuclease-Free Klenow (USB Corp.; Cleveland,Ohio, US); capture beads such as 1 micron streptavidin coatedparamagnetic beads; bead wash buffer comprising for example 0.5MTris-HCL, 0.1M MgCl2, and 10 mM DTT; labeled miRNA elution buffercomprising for example formamide; a buffer exchange device such asMicrocon® YM 10 devices and 0.1× TE wash buffer.

All features disclosed in the specification, including the abstract anddrawings, and all the steps in any method or process disclosed, may becombined in any combination, except combinations where at least some ofsuch features and/or steps are mutually exclusive. Each featuredisclosed in the specification, including abstract and drawings, can bereplaced by alternative features serving the same, equivalent or similarpurpose, unless expressly stated otherwise. Thus, unless expresslystated otherwise, each feature disclosed is one example only of ageneric series of equivalent or similar features.

The foregoing discussion is by no means limiting and other means ofdetecting the miRNAs labeled and measured by the method of the presentinvention can readily be envisioned such as the detection of theextended population of miRNAs by electrophoresis, or for example by theextension of the miRNAs where the sample itself serves as the solidphase such as tissue sections. The invention is described in more detailby the following description.

Example 1

One embodiment of the present method was performed as follows. First, asample of human total RNA from peripheral blood mononuclear cells waslabeled using the labeling method as disclosed in this disclosure, andsubsequently hybridized to a commercially available miRNA microarrayslide, and the fluorescence of the isolated hybridized probes preparedby the method of this disclosure were measured and the results analyzed.

Small RNA Capture-Extension Probe

A set of capture-extension probes comprised miRNA binding segmentscorresponding to 210 unique human miRNAs and 2 replicates of 3additional human miRNAs for a total of 216 capture-extension probes weredesigned to be completely and specifically complimentary to theircorresponding human miRNAs, eight (8) examples of which are depicted inTable III with their different miRNA binding segments, with theirassociated identical solid phase binding segments facilitated byaddition of a biotin attached to the 5′ end of each capture-extensionprobe, their identical extension segments, and their identical spacersegments. The small RNA capture-extension probes were obtained fromIntegrated DNA Technologies, (Coralville, Iowa, US). The individualsmall RNA capture probes were resuspended in 0.1×TE buffer with 2%Acetonitrile (Sigma Aldrich; St. Louis, Mo., US) at a finalconcentration of each probe complex of 100 pmol/μl.

TABLE VII REPRESENTATIVE EXAMPLES OF 5′BIOTINYLATED CAPTURE PROBES AND THEIR COMPLEMENTARY MIRNA CAPTURE PROBECOMPLEMENTARY NAME PROBE SEQUENCE miRNA SEQ ID NO: EPD 1/5BIO/ATTTAGGTGACACTATAGAAACTATACAAC hsa-let-7a SEQ ID NO: 42C TACTACCTCACCCTATAGTGAGTCGTATTA EPD 5/5BIO/ATTTAGGTGACACTATAGAACTATACAACCT hsa-let-7e SEQ ID NO: 43CCTACCTCACCCTATAGTGAGTCGTATTA EPD 14/5BIO/ATTTAGGTGACACTATAGAGCTACCTGCAC hsa-miR-106a SEQ ID NO: 44T GTAAGCACTTTTCCCTATAGTGAGTCGTATTA EPD 24/5BIO/ATTTAGGTGACACTATAGACGCGTACCAAA hsa-miR-126* SEQ ID NO: 45A GTAATAATGCCCTATAGTGAGTCGTATTA EPD 35/5BIO/ATTTAGGTGACACTATAGATCACATAGGAA hsa-miR-135a SEQ ID NO: 46T AAAAAGCCATACCCTATAGTGAGTCGTATTA EPD 39/5BIO/ATTTAGGTGACACTATAGAGATTCACAACA hsa-miR-138 SEQ ID NO: 47C CAGCTCCCTATAGTGAGTCGTATTA EPD 56 /5BIO/ATTTAGGTGACACTATAGACGAAGGCAACAhsa-miR-154 SEQ ID NO: 48 CGGATAACCTACCCTATAGTGAGTCGTATTA EPD 201/5BIO/ATTTAGGTGACACTATAGAAATAGGTCAAC hsa-miR-154* SEQ ID NO: 49C GTGTATGATTCCCTATAGTGAGTCGTATTA “/5BIO” represents a 5′ biotin added insynthesis.

A pool of the 216 capture probes from the Sanger Center was made byadding 7 μl of each probe into one 1.5 ml screw cap tube (Starstedt;Newton, N.C., US). The probes in the mixture were equimolar with respectto one another at a final concentration of approximately 0.5 pmol/eachcapture probe/μl present in the capture-extension probe mixture.

Hybridization of Small RNA to Pooled Capture Probes

Hybridization was carried out by adding 0.5 μg of total RNA isolatedfrom peripheral blood mononuclear cells (BioChain; Hayward, Calif., US)to 1.0 μl of the pooled capture probes at a concentration of 0.5pmol/probe/μl and 10 units of RNasin® (Promega; Madison, Wis., US). Thecomponents were assembled in Bio-Rad 96-well Multiplate (Bio-RadLaboratories, Inc.; Hercules, Calif., US) and briefly pulsed in acentrifuge to mix. Hybridization was performed by incubating thecomponents on a thermocycler (Bio-Rad Laboratories, Inc.) at 42° C. for30 minutes followed by a 1° C. per second decrease to 25° C. for 5minutes.

Labeling by Extension

Using the capture probe as a template and the hybridized miRNA as aprimer, labeling by extension was carried out using KlenowExonuclease-Free DNA Polymerase (USB; Cleveland, Ohio, US). Reactioncomponents containing 1× Klenow Reaction Buffer (USB), 0.1 mMdATP,dGTP,dCPT (Promega Corp.; Madison, Wis., US) with 0.0065 mM Cyanine3-dUTP (Enzo Life Sciences, Inc.; Farmingdale, N.Y., US) and 5 unitsKlenow Exonuclease Free DNA Polymerase (USB) were mixed and then addedto the wells containing the hybridized miRNA and capture probes for atotal of 10 μl reaction volume. The plate was then briefly pulsed in acentrifuge to mix. The plate was covered in foil to protect in fromlight and incubated at room temperature for one hour. The reaction wasstopped by adding 1.0 μl of 0.5M EDTA (Sigma Aldrich Corp.; St. Louis,Mo., US). Electrophoresis was performed using 1.0 μl of the labelingreaction product run on precast Nuseive/GTG 3:1 agarose gels containingethidium bromide (BMA CORP.; Rockland, Me., US). The labeling wasconfirmed by observation of a cluster of bands of the appropriate sizefor the family of extension products.

Magnetic Bead Capture and Elution Labeled Small RNA from the CaptureProbe

The remaining 9 μl of the labeling reaction mix was transferred into anew 1.5 ml screw cap tube (Starstedt; Newton, N.C., US) and 5.0 μl ofStreptavidin A-Beads™ (Aureon; Vienna, Austria). The tubes were flickedto mix and incubated to capture the probe-miRNA extension product for 20minutes at room temperature using foil to protect it from lightexposure. After 20 minutes the bead-probe hybrid complex were washed byplacing the 1.5 ml tube on a magnet stand (Grace Biolabs; Bend, Oreg.,US), and allowing the beads collect at side of tube. The excess liquidwas pipetted off and discarded followed by the addition of 100 μl of 1×Klenow Exonuclease Free reaction buffer (USB). The tube was flicked tomix the contents and then placed back onto the magnet stand, excessliquid was drawn off and discarded, and 100 μl of 1× Klenow ExonucleaseFree reaction buffer (USB) was added. The tube was mixed, then placedback onto the magnet stand, excess liquid pipetted off and discarded and20 μl of 100% Deionized Formamide (BioVentures, Inc.; Murfreesboro,Tenn., US) added to the tube to elute the labeled small RNA extensionproduct from the capture probe-bead complex. Two (2) minutes after theformamide was added, the tube was flicked to mix and placed back ontothe magnet stand. The 20 μl of formamide containing the eluted labeledsmall RNA extension product was transferred into a new 1.5 mL screw captube. A buffer exchange was then carried out to replace the formamidewith 0.1× TE using a Microcon® YM 10 ultrafiltration device (MilliporeCorp.; Billerica, Mass., US). 180 μl of 0.1× TE was added to the 20 μlvolume containing the eluted labeled small RNA extension product and theentire volume, 200 μA was transferred to the column placed in a 1.5 mlcollection tube. The tube was then spun at 14,000×g in a centrifuge at10° C. for 30 minutes. The flow through was discarded and theultrafiltration device placed back into the collection tube and 100 μlof 0.1× TE added to the ultrafiltration device. The ultrafiltrationdevice was then spun again at 14,000×g for 10 minutes at 10° C. and theflow through discarded. The ultrafiltration device was then inverted andplaced in a new collection tube and backspun at 3,000×g for 5 minutes at10° C. The 20 μl of recovered volume contained the labeled small RNA ina suitable buffer for hybridization.

Hybridization of Labeled Small RNA Onto Small RNA BioChip

25 μl of 2× Hybridization buffer (Genosensor; Tempe, Ariz., US) wasadded to the 20 μl of labeled miRNA and 5 μl of sterile DI water,briefly pulsed to mix and pipetted onto the active area of theGenoExplorer™ small RNA BioChip (Genosensor) and covered with a 20×20plastic coverslip (TedPella, Inc.; Redding, Calif., US). The slide wasthen placed in a humid chamber, sealed and placed in the dark at roomtemperature to hybridize overnight for approximately 18 hours. After theovernight incubation, the slide was washed in once in 25 ml of 3×SSCbuffer and 0.2% SDS for 5 minutes followed by a second wash in 25 ml of1×SSC and 0.1% SDS for 5 minutes. A third and fourth wash were doneusing 0.1×SSC buffer for 2 minutes each. Washing consisted of immersingthe slide in the buffer and gentle agitation for the stated amount oftime. After washing the slides were air dried with compressed airfollowed by slide scanning.

Slide Scanning and Analysis

The slide was scanned using a ScanArray® Express HT Microarray Scannerby Perkin Elmer (Wellesley, Mass., US). An easy scan of the full slideat 70% gain and 20 μm resolution was sufficient for quantitation by theinstrument software. Spot finding and slide information was completed byimporting the .gal file provided by the manufacturer of the miRNAmicroarray. The .gal file included all spot identified as well aslocation, diameter and spacing of the slide spots.

TABLE VIII REPRESENTATION OF THE INPUT DATA FOR QUANTITATION BEGIN ARRAYPATTERN INFO Units μm Array Rows   4 Array Columns   1 Spot Rows  21Spot Columns  24 Array Row Spacing 5000 Array Column Spacing 5000 SpotRow Spacing  200 Spot Column Spacing  200 Spot Diameter  120Interstitial   0 Spots Per Array  504 Total Spots 2016 END ARRAY PATTERNINFO BEGIN IMAGE INFO ImageID Channel Image Fluorophore −1 CH1 Alexa 546−1 CH2 Alexa 546 END IMAGE INFO BEGIN NORMALIZATION INFO NormalizationMethod   LOWESS END NORMALIZATION IFO

Referring now to FIG. 8, there is shown a scanned image of thehybridized GenoExplorer™ miRNA chip using 10 μm scan resolution and a70% laser gain.

According to the miRNA microarray slide layout, each spot in printed intriplicate allowing for spot to spot normalization within each spot, aswell as across the entire slide. In addition to miRNA spots, there werenegative control spots consisting of either buffer only or other unknownsequences that should not fluoresce with the addition of sample. TheGenoExplorer™ miRNA chip contains a total of 632 spots representingmature and precursor miRNAs in triplicate. The mean and medianintensities of all the negative control spots were averaged to give arepresentative value for background levels. Each mean and medianintensity value of the background was subtracted from the mean andmedian intensity value of the sample spots to give a signal minus noisevalue. Once obtained, the individual sample values were averaged acrossthere triplicate values to give an average signal intensity value foreach represented sample spot. Those sample spots with high standarddeviations between the triplicate spots were not used for final analysisas they did not accurately represent the sample intensity. Overall 198of the 216 capture probes that were added to the total RNA sample forsmall RNA extension labeling were considered positive once normalized.The log base 2 (log 2) or signal intensities after normalization andranged from a positive low of 4.64 to a positive high of 13.28. ThemiRNA, hsa-mir-198, corresponded to the highest signal intensity of log2 13.28 with a spot to spot standard deviation of log 2 0.099.Additional spots were present but thrown out due to one of thetriplicate spots lying outside an acceptable intensity range compared tothe other triplicate spots.

Referring now to FIG. 9, there is shown a graph of fluorescent intensityfor the sample of 12 different miRNAs detected in 0.5 μg of human totalRNA isolated from peripheral blood mononuclear cells using cyanine 3,where the 12 different miRNAs detected were from 1 to 12: 135a, 369,024, 453, 154, 7e, 154*, 138, 325, 106a, 126 and 7a.

The specific of miRNAs represented in peripheral blood mononuclear cellswas unknown before conducting this experiment. Most miRNA microarraystudies are performed on specific tissue such as tumor and non-tumor.The results of this microarray show that using the method of the presentinvention the capture-extension probes can specifically capture andfacilitate labeling the miRNAs present in a total RNA sample and thelabeled product is useful for downstream applications such as microarrayanalysis. In this example a relatively small sample size of 0.5 μg oftotal RNA was used and resulted in acceptable signal intensities whenhybridized onto a microarray. Most current labeling methods require asmuch as 50 μg of starting total RNA material in order to receiveacceptable signal intensity levels after slide hybridization.

Example 2

To illustrate the nick-amplification method the following experiment wasperformed. In brief, synthetic microRNAs were amplified, detected andanalyzed using the method and reagents described below.

Small RNA Nick-Amplification Probes

A small set of nick-amplification probes was designed to be completelyand specifically complementary to a set of human microRNAs (miRNAs). Theset of nick-amplification probes was obtained from Integrated DNATechnologies, (Coralville, Iowa, US). Each of the individual probes wasre-suspended in 0.1×TrisEDTA (Sigma Aldrich: St. Louis, Mo., US) with 2%acetonitrile (Sigma Aldrich) at a final concentration of 100 pmol/μl.

COMPLEMENTARY NICK-AMP PROBE NAME PROBE SEQUENCE 5′-3′ miRNA SEQ ID NO:34 AMP dU_hsa-miR-135a ATCAGGA/ideoxyU/CAGCTGA hsa-miR-135aSEQ ID NO: 24 GCCTCAGCATTCACATAGGAA TAAAAAGCCATAACAC38 AMP dU_hsa-miR-138 ATCAGGA/ideoxyU/CAGCTGA hsa-miR-138 SEQ ID NO: 25GCCTCAGCATGATTCACAACA CCAGCTACAC 56 AMP dU_hsa-miR-154ATCAGGA/ideoxyU/CAGCTGA hsa-miR-154 SEQ ID NO: 26 GCCTCAGCATCGAAGGCAACACGGATAACCTAACAC

Synthetic MicroRNAs

The synthetic miRNA (syn-miRNA) were selected and designed tospecifically reflect a set of human miRNAs. The syn-miRNAs were obtainedfrom Integrated DNA Technologies (Coralville, Iowa, US). The syn-miRNAswere resuspended in stabilization buffer containing 1 mM Sodium Citrate(Ambion; Austin, Tex., US) and 30% Formamide (Bioventures; Murfreesboro,Tenn., US) at a final concentration of 100 pmol/μl. The syn-miRNAs werethen aliquoted into 10 μl working stocks in 0.5 ml tubes (Nalgene;Rochester, N.Y., US) to reduce freeze thaw effects.

SYNTHETIC MICRORNA HUMAN SEQ ID NAME RNA SEQUENCE 5′-3′ (rN =ribonucleotide) miRNA NO: hsa-miR-/5Phos/rArGrCrUrGrGrUrGrUrUrGrUrGrArArUrC hsa-miR- SEQ ID  138 138NO: 50 hsa-miR- /5Phos/rUrArUrGrGrCrUrUrUrUrUrArUrUrCrCrUrArUrGrUrGrAhsa-miR- SEQ ID  135a 135a NO: 51 hsa-miR-/5Phos/rUrArGrGrUrUrArUrCrCrGrUrGrUrUrGrCrCrUrUrCrG hsa-miR- SEQ ID  154154 NO: 52

Quencher/Fluor Probes

A quencher/fluor (QF) probe was designed to be recognized and nicked bynicking enzyme Nb.BbvC1A. The 51AbFQ refers to a 5′ Iowa Blackfluorescent quencher that will quench the FAM fluorescence until theprobe is nicked or cut by one or more suitable enzyme. The QF probe wasobtained from Integrated DNA Technologies, (Coralville, Iowa, US). Theprobe was resuspended in 0.1× TE with 2% acetonitrile (Sigma Aldrich:St. Louis, Mo., US) at a final concentration of 100 pmol/μl.

NICKING Q-F PROBE ENZYME NAME PROBE SEQUENCE 5′-3′ SITE SEQ ID NO:QF PROBE 2 5′_3′ 5′/5IAbFQ/CAGGATCAGCTGAGAGC Nb.BbvCI SEQ ID NO: 31CTCA/36-FAM/3′

Nick Extension and Amplification Reaction

All reaction components were thawed and assembled on ice to minimize anyenzyme activity before the desired reaction start time and ortemperature. Six (6) different reactions with four (4) duplicates ofeach reaction were assembled using the following master mix: 1×NEBBuffer 2 (New Enland Biolabs; Ipswich, Mass., US), 50 μM dNTP mix (eachdA, dC, dG, dT, (Bioventures, Inc.)), 0.05 pmol of syn-miRNA, 1 pmol ofNick-Amp Probe, 1 pmol of QF Probe, 2 units of Exonuclease-Free Klenow(USB Corporation; Cleveland, Ohio, US), 1 unit of Nt.AlwI enzyme (NewEngland Biolabs), 1 unit of Nb.BbvCI (New England Biolabs) and sterilenuclease-free water to a final volume of 20 μl per reaction. The 6reactions differed in their syn-miRNA and Nick-Amp probe combinationaccording to the following matrix; all reactions contained the same QFprobe:

syn-miRNA Name Nick-Amp Probe Name REACTION 1 hsa-miR-135a34 AMP dU_hsa-miR-135a REACTION 2 None 34 AMP dU_hsa-miR-135a REACTION 3hsa-miR-138 38 AMP dU_hsa-miR-138 REACTION 4 None 38 AMP dU_hsa-miR-138REACTION 5 hsa-miR-154 56 AMP dU_hsa-miR-154 REACTION 6 None56 AMP dU_hsa-miR-154

Once all reactions were assembled on ice, 20 μl was then pipetted intothe wells of pre-chilled 0.2 ml low profile white strip tubes (Bio-Rad;Hercules, Calif., US) strips. The strips were then capped with 0.2 mlclear flat cap strips from Bio-Rad and placed on a Chromo4 Real Time PCRinstrument (BioRad). The reactions were performed using the followingprogram programmed using Opticon Monitor2 software (Bio-Rad): Step 1: 5minute hold at 4° C., plate read, Step 2: 37° C. for 30 seconds, plateread, Step 3: go to Step 2 99 more times, Step 4: plate read, End. Uponcompletion of the program the data was analyzed as follows.

Nick Amplification Analysis

Opticon Monitor2 software was used to analyze the results of theNick-Amplification reactions. A manual threshold of 0.04 fluorescencewas set to allow for any initial fluorescence that was present beforethe reactions started. The results show that for all reactions withsynthetic microRNAs present in the reaction, the fluorescence signalincreased above background at 2 to 3.9 times the C(t) of the no microRNAreactions. The chart below gives the average cycle count, C(t), acrossthe 4 replicates for each reaction, at which each reaction signalcrossed above the set threshold value (0.04 Fluorescence).

WELL RANGE REACTION NAME SYN-MIRNA NICK-AMP PROBE C(t) AVERAGE C1-F1Reaction 1 hsa-miR-135a 34 AMP dU_hsa-miR-135a 39.95 C3-F3 Reaction 2None 34 AMP dU_hsa-miR-135a 79.91 C5-F5 Reaction 3 hsa-miR-138 38 AMPdU_hsa-miR-138 17.58 C7-F7 Reaction 4 None 38 AMP dU_hsa-miR-138 50.87C9-F9 Reaction 5 hsa-miR-154 56 AMP dU_hsa-miR-154 24.98 C11-F11Reaction 6 None 56 AMP dU_hsa-miR-154 99.08

The present invention has been discussed in considerable detail withreference to certain preferred embodiments, other embodiments arepossible. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure. All references cited herein are incorporated by reference intheir entirety.

What is claimed is:
 1. A capture probe comprising a polynucleotide, the polynucleotide comprising: (a) a spacer segment comprising a spacer segment sequence, the spacer segment comprising a 3′ end and a 5′ end; (b) a template segment comprising a template segment sequence, the template segment comprising a 3′ end and a 5′ end; and (c) a small polynucleotide binding segment comprising a small polynucleotide binding segment sequence; wherein the small polynucleotide binding segment is substantially complementary to, and capable of hybridizing to, one or more than one small polynucleotide of interest by Watson-Crick base pairing, wherein the small polynucleotide of interest is selected from the group consisting of a RNA polynucleotide, a DNA polynucleotide and a combination thereof; wherein the 5′ end of the spacer segment is connected to the 3′ end of the small polynucleotide binding segment; and wherein the 3′ end of template segment is connected to the 5′ end of the small polynucleotide binding segment.
 2. The capture probe of claim 1, further comprising a solid phase binding segment of a molecular composition capable of binding to a solid phase.
 3. The capture probe of claim 1, wherein the spacer segment includes a RNA polymerase termination site.
 4. The capture probe of claim 1, wherein the small polynucleotide binding segment is substantially complementary to, and capable of hybridizing to, a miRNA of interest.
 5. The capture probe of claim 1, wherein the template segment includes one or more than one sequence selected from the group consisting of: (a) a polynucleotide polymerase recognition site or a sequence that is complementary to a polynucleotide polymerase recognition site; (b) one or more than one sequence that is a restriction enzyme recognition motif; and (c) one or more than one sequence that is complementary to a RNA-cleaving catalytic nucleic acid.
 6. A composition comprising two or more capture probes according to claim 1, the composition comprising: (a) a first capture probe comprising a first spacer segment, a first small polynucleotide binding segment and a first template segment; and (b) a second capture probe comprising a second spacer segment, a second small polynucleotide binding segment and a second template segment, where the second small polynucleotide binding segment comprises a different polynucleotide binding segment sequence than the first polynucleotide binding segment and the second template segment comprises a different template segment sequence than the first template segment. 